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Alterations of adrenomedullin and its receptor system components in calcified vascular smooth muscle cells
Regulatory Peptides 120 (2004) 77 – 83 www.elsevier.com/locate/regpep
Alterations of adrenomedullin and its receptor system components in calcified vascular smooth muscle cells Chun Shui Pan a, Yong Fen Qi a,b,c,*, Shu Heng Wang b, Jing Zhao b, Ding Fang Bu a, Gui Zhong Li a, Chao Shu Tang a,c b
a Institute of Cardiovascular Diseases, Peking University First Hospital, Beijing 100034, China Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100083, China c Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100083, China
Received 7 November 2003; received in revised form 18 February 2004; accepted 25 February 2004
Abstract Vascular calcification is a common finding in many cardiovascular diseases. Paracrine/autocrine changes in calcified vessels, and the secreted factors participate in and play an important role in the progress of calcification. Adrenomedullin (ADM) is a potent vasodilator peptide secreted by vascular smooth muscle cells (VSMCs) and vascular endothelial cells. Recently, receptor activity-modifying proteins (RAMPs) have been shown to transport calcitonin receptor-like receptor (CRLR) to the cell surface to present either as CGRP receptor or ADM receptor. In this work, we explored the production of ADM, alterations and significance of ADM mRNA and its receptor system components—CRLR and RAMPs mRNA in calcified VSMCs. Our results showed that calcium content, 45Ca2 + uptake and alkaline phosphatases (ALPs) activity in calcified VSMCs were increased, respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% ( p < 0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified cells were elevated, respectively, compared with that of control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA were significant correlation with ADM mRNA (r = 0.83, 0.92 and 0.93, respectively, all p’s < 0.01) in calcified VSMCs. The results show that calcified VSMCs generate an increased amount of ADM, up-regulate gene expressions of ADM and its receptor system components—CRLR, RAMP2 and RAMP3, suggesting an important role of ADM and its receptor system in the regulation of vascular calcification. D 2004 Elsevier B.V. All rights reserved. Keywords: Calcification; Vascular smooth muscle cells (VSMCs); ADM; CRLR; RAMPs
1. Introduction Vascular calcification is a common finding in many cardiovascular diseases, such as hypertension, atherosclerosis, diabetes, chronic renal failure, aging, aortic stenosis and prosthetic valve replacements [1 –3]. Calcified vessel causes a decreased function of vasodilatation, increased stiffness and promoted form of thrombus and atherosclerotic plaques rupture. Vascular calcification was believed to be an important risk factor of cardiovascular diseases [4,5]. However,
* Corresponding author. Department of Physiology and Pathophysiology, Institute of Cardiovascular Diseases, Peking University Health Science Center, Beijing 100083, China. Tel.: +86-10-82802183; fax: +86-1066556255. E-mail address: [email protected] (Y.F. Qi). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.02.020
the mechanism by which vascular calcification causes vascular dysfunction and remodeling is not clear yet [6,7]. Previously, calcification was generally considered as a passive calcium deposition in extracellular matrix and cells. The view, however, has changed in recent years, and the vascular calcification is considered as an active, regulative process similar to the osteogenesis [8]. During vascular calcification, the various vascular cells, including vascular smooth muscle cells (VSMCs), pericytes, fibroblasts and macrophages, transformed into osteoblast-like phenotype, which was characterized by an increase in alkaline phosphatase (ALP) activity, matrix vesicle formation and overexpression of bone morphogenetic proteins (BMPs) including BMP-2 and bone matrix proteins such as osteopontin, osteonectin, osteocalcin, etc. [5,9 – 11]. However, the changes in function of the vascular cells with altered phenotype and its pathophysiological significance remain
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unclear. It is common knowledge that paracrine/autocrine factors secreted from vascular cells contribute to circulatory homeostasis, and mediate pathogenesis of cardiovascular diseases. We supposed that phenotype-altered vascular cells not only had some properties of ossification, but also changed their paracrine/autocrine functions, and that the paracrine/autocrine dysfunction of calcified vascular vessels might play an important role in calcification-induced vascular damage. Adrenomedullin (ADM) was initially isolated from human pheochromocytoma cells by Kitamura et al. [12] in 1993 as a peptide capable of a potent and long-lasting hypotensive effect. ADM in blood circulation is mainly synthesized and secreted from vascular endothelial and smooth muscle cells. ADM has been shown to have vasodilatory, hypotensive and growth regulating properties, and is considered as an important regulatory peptide contributed to vascular homeostasis [13,14]. It was wellknown that ADM levels in plasma and cardiovascular tissues were compensatively elevated during cardiovascular diseases, as hypertension, heart failure, atherosclerosis, etc. [13,15]. Our previous researches found that ADM production was increased, and ADM gene was upregulated in calcified VSMCs in vitro and in calcified vessels of rat in vivo. Administration of ADM attenuated significantly pathogenesis of vascular calcification induced by vitamin D and nicotine [16,17]. However, it is unclear yet whether ADM receptor in calcified vessels changes or not, and which role do these changes play in pathogenesis of vascular calcification. It is well-known that VSMCs is highly responsive to ADM and rich in ADM receptors [18]. Recent researches showed that receptors bound with ADM were orphan L-1 and RDC-1 receptors [19]. In addition, another orphan receptor named calcitonin receptor-like receptor (CRLR) could bind with ADM when receptor activity-modifying protein (RAMP)2/RAMP3 existed on cellular membrane surface [20]. To facilitate the characteristics of alterations of autocrine and/or paracrine function in calcified vascular cells, we developed an in vitro calcification system in which diffused calcification could be induced by culturing rat VSMCs in the presence
of h-glycerophosphate. In this study, we observed the changes in ADM, and its receptor system components— CRLR and RAMP2/3 in calcified VSMCs to illustrate the role of ADM/ADM receptor pathway in pathogenesis of vascular calcification.
2. Materials and methods 2.1. Animals and reagents All animal experiments in this study were performed with the approval of the Animal Care Committee of the First Hospital, Peking University. Rat radioimmunoassay (RIA) kit for ADM was provided by Phoenix Pharmaceutical (St. Joseph, MO). h-Glycerophosphate and ALP assay kit were purchased from Sigma (St. Louis, MO). 45CaCl2 was obtained from NEN Life Sci Products (Boston, MA, USA) and Trizol was purchased from GIBICO. dNTP was purchased from Clontech. M-MuLV reverse transcriptase, Taq DNA polymerase, RNasin, Oligo(dT)15 Primer were provided by Promega. Other chemicals and reagents were of analytical grade. Sequences of oligonucleotide 10 primers for amplification are synthesized by SBS (China), as shown in Table 1. 2.2. Cell culture of VSMCs The explant culture method of VSMCs was described previously [21]. Briefly, rat thoracic aorta was cut into small pieces (about 2 – 3 mm each) after partial removal of external connective tissues, placed in DMEM medium supplemented with 4.5 g/l glucose, 10 mM sodium pyruvate and 20% fetal bovine serum and incubated at 37jC in an incubator containing 95% air and 5% CO2. VSMCs migrated from the explants were collected and maintained in growing medium (DMEM containing 10 mM sodium pyruvate and 15% fetal bovine serum). The a-actin examination of cultured cells confirmed a positive response. VSMCs of 5th to 8th passages were used for the experiments.
Table 1 Oligonucleotides used for the amplification Target ADM CRLR RAMP2 RAMP3 h-Actin
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Sequence
Size (bp)
Ann.T (jC)
Cycle
5V-CTCGACACTTCCTCGCAGTT-3V 5V-GCTGGAGCTGAGTGTGTCTG-3V 5V-CAACTGCTGGATCAGCTCAG-3V 5V-CATCGCTGATTGTTGACACC-3V 5V-TGAGGACAGCCTTCTGTC A-3V 5V-CATCGCCGTCTTTACTCC TC-3V 5V-CTTCTCCCTCTGTTGCTGCT-3V 5V-CACAGAAGCCGGTCAGTGT-3V 5V-ATCTGG AC CAC ACC TTC-3V 5V-AGCCAG GTC CAG ACG CA-3V
446
61
30
446
55
28
371
55
28
416
57
32
291
55
24
Expected sizes of the amplicons, annealing temperature (Ann.T) used for the PCR reaction.
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2.3. Cell calcification and assay of calcification ( 45Ca accumulation) [10] After confluence, the cells were inoculated on 24-well plates (104 cells/ml) in DMEM containing 10 mM sodium pyruvate supplemented with 15% FBS in the absence (i.e., growing medium, as control group) or in the presence of 10 mM h-glycerophosphate (i.e., calcification medium, as calcification group). The medium was replaced with fresh medium every 3 days. The treatment continued for 14 days. Twenty-four hours before the end of treatment, 1.0 ACi/ ml of 45CaCl2 was added in the medium. After 24 h of incubation, the medium was removed and the cell layer was washed five times with cold PBS, scraped into borosilicate tubes containing 0.5 ml of perchloric acid, and spun vigorously. Then, 0.5 ml of H2O2 was added and the suspensions were incubated for 60 min at 80 jC. After incubation, the mixture was dissolved in 1.0 ml of ethylene glycol monoethyl ether and spun vigorously, and radioactivity was measured by h-Liquid Scintillation Counting (Beckman, LS 6500). 2.4. Assay of cellular calcium content [10] After the end of incubation, the medium was removed and the cell layer was washed five times with cold PBS. Scraped cells were collected, and dissolved in HNO3 and diluted with a blank solution (27 nM KCl, 25 AM LaCl3), the calcium content was measured on an atomic absorption spectrophotometer at 422.7 nm. 2.5. Assay of AP activity [10] A cell-associated ALP activity assay was performed with a modification of the ALP assay kit from Sigma. The cells were rinsed three times with cold PBS and scraped into 200 Al of lysis buffer (0.2% NP-40 in 1 mM MgCl2) with a rubber policeman and sonicated for 10 s. Next, 1 ml of reaction mixture was added to each well. Reaction mixture was 221 alkaline buffer: stock substrate solution 1:1. Stock substrate solution was prepared by dissolving the contents of a 100-mg capsule of Sigma 104-phosphatase substrate in 25 ml of ddH2O. This mixture was then incubated for 30 min at 37 jC. The yellow color was indicative of ALP activity. The reaction was stopped by the addition of 12 Al of 1 M NaOH to each well, and absorbance was determined at 405 nm on spectrophotometer. ALP activity was calculated using U-nitrophenol as a standard, according to the kit’s instruction (Sigma). One unit was defined as the activity producing l nmol of U-nitrophenol for 30 min. 2.6. von Kossa calcium staining [22] Cultured cell monolayers were fixed in 0.1% glutaraldehyde in PBS for 30 min at room temperature. Cells were
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then washed twice with double-distilled water and incubated with 5% silver nitrate for 30 min at room temperature in the dark. The cells were washed gently with double-distilled water, exposed to ultraviolet light for 30 min, and counterstained with 0.1% eosin for 30 s. Calcium mineral stained black. Calcified nodules in each well were countered twice by use of phase-contrast microscope by two different individuals blinded to treatment conditions. 2.7. Radioimmunoassay for ADM [23] One milliliter of the medium at the end of cell culture was collected and immediately acidified with acetic acid to a final concentration of 1.0 M. The acidified medium was heated at 100 jC for 10 min to inactivate proteases, and applied to a Sep-Pak C18 cartridge (Millipore-Waters, Milford, MA, USA). After washing the cartridge with 10% CH3CN in 0.1% trifluoroacetic acid, the absorbed materials were eluted with 50% CH3CN in 0.1% trifluoroacetic acid, lyophilized and stored at 30 jC. The immunoreactive-ADM (ir-ADM) in the medium extract was measured using a specific RIA kit for ADM. Rabbit polyclonal anti-ADM was used for RIA in this kit. The IC50 of rat ADM was 13– 47 pg per tube, and the crossreactivity with rat ADM was 100%, and was zero with proadrenomedullin N-terminal 20 peptide, amylin and endothelin. Secretion of ADM from VSMCs was presented as fmol/105 cells. 2.8. Characterization of secreted ADM [24] To examine the molecular forms of ir-ADM, the extract of conditioned medium was analyzed by reverse-phase high-performance liquid chromatography (HPLC) with a column of TSK ODS 120 A (4.6 mm 25 cm, Tosoh, Tokyo, Japan). The extracts were eluted from the column with a linear gradient of 0 – 60% acetonitrile in 0.1% trifluoroacetic acid, and the ir-ADM in each fraction was measured with the RIA. The recovery of ir-ADM in the HPLC was greater than 80%. 2.9. Measurement of ADM, CRLR, RAMP2 and RAMP3 mRNA Total RNA of VSMCs was prepared by in situ lysis of the cells using Trizol (Gibco-BRL, Rockville, MD) reagent after washing the cells with ice-cold phosphate buffer solution. One microgram of total RNA was reverse-transcribed into single strand cDNA using M-MuLV reverse transcriptase and oligo(dT)15 primer. PCR was performed in a 0.2-ml tube containing VSMC cDNA 2 Al, 5 AM per each ADM-S and ADM-A primer mixture 1 Al, 2.5 mM per each dNTPs mixture 1 Al, 1.5 mM MgCl2 1.5 Al, 10 PCR buffer 2.5 Al and 1.25 unit Taq DNA polymerase, in a total volume of 25 Al. After denaturing at 95 jC for 5 min, PCR was run at 94 jC 30 s, 61 jC 30 s and 72
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2.10. Statistical analysis The values of various parameters were averaged from six independent experiments with duplication. Results of some experiments were normalized to total protein determined by Bradford’s method. The data were expressed as means F S.D. Comparisons across two groups were made using Student’s t test. Linear regression analysis was used to assess the correlation between variables. A p value < 0.05 was considered statistically significant.
3. Results 3.1. The general characteristics of VSMCs calcification Calcium mineral deposits formed by cultured VSMCs were assessed by the silver nitrate method (von Kossa staining). In calcified VSMCs, formation of multicellular nodules was shown. von Kossa staining for calcification, showed positive staining as black/brown areas within the main, large, nodular structures as shown in extracellular matrix and cytoplasma (Fig. 1). The content of calcium, 45 Ca2 + uptake and ALP activity in calcified VSMCs were increased by 118%, 174% and sevenfold (all p’s < 0.01), respectively, compared with that of control. (Table 2). 3.2. Increase in ADM secreted by calcified VSMCs and characteristic of secreted ADM
Fig. 1. (A) Calcification of multicellular nodules. von Kossa staining for calcification, showing positive staining as black/brown areas within the main, large, nodular structure (original magnification 40 ). (B) Formation of multicellular nodules in culture phase contrast microscopy showing rat VSMCs (original magnification 20 ).
jC 40 s for 30 cycles, 72 jC 5 min. Six microliters of PCR product was separated in a 1.5% agarose gel, and stained with ethidium bromide. The optical density of the 446-bp band was measured using the Gel Documentation System (Bio-Rad, Hercules, CA). Amplification of ADM cDNA was confirmed by digestion of the PCR products with restriction enzyme BglII. To calibrate the sample loaded in quantitative PCR, h-actin cDNA was determined at the same time. Two microliters of PCR product was amplified again using the two rat h-actin primers at 94 jC 30 s, 55 jC 30 s, and 72 jC 40 s for 24 cycles, 72 jC 5 min, and the optical density of the h-actin band (291 bp) was measured. The ratio of ADM mRNA/h-actin mRNA is the relative amount of ADM mRNA. The relative amount of CRLR, RAMP2, RAMP3 mRNA was determined according to the above method.
Content of ADM in calcified VSMCs medium was obviously increased by 99% as compared with control (17.82 F 1.68 versus 8.97 F 0.61 fmol/ml, p < 0.01). Molecular forms of ADM secreted into the medium were characterized by reverse-phase HPLC (Fig. 2). The irADM was composed of one major and one minor peak, and the major peak appeared at an elution position identical to that of synthetic rat ADM (1 – 50)-NH2, a whole active molecule of the rat ADM peptide. 3.3. Up-regulation of ADM mRNA in calcified VSMCs Semi-quantitative RT-PCR was performed to determine the ADM mRNA level of VSMCs. ADM gene products were identified by restriction enzyme BglII, and it was cut into two fragments, a 156-bp fragment and a 290-bp fragment confirmed that the ADM RT-PCR product was Table 2 General index of calcified VSMCs (n = 6, X F S.D.)
Control Calcified VSMC
45 Ca uptake (103 cpm/mg Pr)
Ca content (nmol/mg Pr)
ALP activity (U/mg Pr)
32.2 F 5.20 88.2 F 10.21**
46.8 F 7.23 102.2 F 18.23**
74 F 11 624 F 84**
ALP: alkaline phosphatase. ** p < 0.01 vs. control.
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Fig. 2. Analysis of reverse-phase HPLC of immunoreactive ADM (irADM) secreted from VSMCs into the media. A linear gradient of acetonitrile of 10 – 60% trifluoroacetic acid for 60 min at a flow rate of 1.0 ml/min. The arrow indicates the elution position of synthetic rat ADM (1 – 50)-NH2.
correct. The ADM mRNA level in calcified VSMCs was elevated by 78% ( p < 0.01) compared to control group. Results are shown in Fig. 3. 3.4. Up-regulation of ADM receptor system components mRNA in calcified VSMCs According to the methods described above we obtained the RT-PCR products of the CRLR, RAMP2 and RAMP3. These products were proved by the sequence analysis using the respectively relevant primer, which testified the RT-PCR products of CRLR, RAMP2 and RAMP3 were correct. The relative amounts of CRLR, RAMP2 and RAMP3 mRNA in calcified group were increased by 93.7%, 91.8% and 109.5% (all p’s < 0.01), respectively, compared with control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA
Fig. 4. Alterations of CRLR, RAMP2, RAMP3 mRNA in calcified VSMCs mRNA expression were determined by semi-quantitative RT-PCR analysis. CRLR: calcitonin receptor like receptor. RAMP: receptor activitymodifying protein. (A) RT-PCR products of CRLR, RAMP2 and RAMP3 mRNA were separated in a 1.5% agarose gel. (B) Quantitative analysis of changes in CRLR, RAMP2 and RAMP3 mRNA expression. Bars represent mean F S.D. of three individual samples. **p < 0.01, compared with control.
were in significant correlation with ADM mRNA (r = 0.83, 0.92 and 0.93, respectively, all p’s < 0.01) in calcified VSMCs. Results are shown in Fig. 4.
4. Discussion
Fig. 3. Change of ADM mRNA in VSMCs of rats. mRNA expressions were determined by semi-quantitative RT-PCR analysis. ADM: adrenomedullin. (A) RT-PCR products of ADM mRNA were separated in a 1.5% agarose gel. (B) Quantitative analysis of changes in ADM mRNA expression. Bars represent mean F S.D. of three individual samples. **p < 0.01, compared with control.
Vascular calcification is an active regulatory progress similar to osteogenesis and osteoporosis. Calcified vascular cells with osteoblast-like cell phenotype traits are cellular basis of vascular calcification pathogenesis [8]. The VSMCs transformed to osteoblast-like cells may not only result in an increase of vascular rigidity, which correlates with calcium phosphate deposition, but also influence vascular autocrine and/or paracrine function, and change vascular responsiveness to vasoactive substances [6,7]. In this study, we used cultured rat VSMCs treated with hglycerophosphate as a model of vascular calcification. hGlycerophosphate is a substrate for ALP. It can release inorganic phosphate to raise the local concentration of phosphorous, accelerate calcium and phosphorous deposition on cells and tissues, and induce formation of calcified nodule [10]. In the calcified VSMCs, we found that the calcium content, 45Ca2 + uptake and ALP activity were significantly increased compared with normal VSMCs. The alterations of these parameters demonstrated that calcified VSMCs had osteoblastic-like characteristics, which were in accordance with previous research [10]. It is known that VSMCs produce and secrete ADM, but cannot store it.
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The ADM level in the medium was generally considered as the amount of ADM synthesized by VSMCs [23]. In this work, we found that content of ADM in the medium was higher in calcified VSMCs than that of control VSMCs, and the level of ADM mRNA was also higher in calcified VSMCs than that of controls. The data show that the increase of ADM production in calcified VSMCs results from up-regulation of ADM gene expression. The ADM gene, located on chromosome 11, consists of four exons and three introns, and contains upstream transcription factor activator protein 2 (AP-2) and cAMP-regulated enhancer sequences [25]. Previous data showed that induction of AP2, secondary to activation of phospholipase C and protein kinase C, might be the mechanism by which growth factors such as fibroblast growth factor and platelet- and epidermalderived growth factors stimulated a high transcription and expression of the ADM gene in VSMCs [14,25]. The mechanism of ADM gene over-expression in calcified VSMCs, however, needs further investigation. A recent report [26] showed that CRLR could function as a calcitonin gene-related peptide (CGRP) receptor when coexpressed with a single transmembrane domain protein known as RAMP1, and as an ADM receptor when coexpressed with either RAMP2 or RAMP3. Interestingly it appears that the different RAMPs influence glycosylation leading to a detectable size difference between CRLR species with CGRP or ADM receptor activity. RAMP2/3 has been shown to modify the posttranslational processing of the CRLR, leading to high-affinity ADM binding [19]. In the present experiment, the data showed that the gene expressions of CRLR, RAMP2 and RAMP3 were obviously increased in calcified VSMCs, and the alterations of those mRNA levels were positive correlation with that of ADM mRNA. It was reported that ADM, CRLR, RAMP2 and RAMP3 mRNA were upregulated in many cardiovascular diseases, such as hypertension, heart failure, artherosclerosis, etc., and that activated ADM/ADM receptor system might play a compensatory protective role in cardiovascular diseases. RAMP2 gene is located on chromosome 17 and is composed of four exons separated by small introns. The first exon encodes 5V UTR sequence, while exon 4 is well-conserved and encode all of the C-terminal and transmembrane domains plus the distal 64 amino acids of N-terminal [18,19]. RAMP3 gene is located on chromosome 7 and comprise at least three exons with a large intron (>17 kb) separating the first two exons and a small (>5 kb) intron separating exons 2 and 3. It was reported that the CRLR genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor and hypoxia-inducible factor-1 alpha (HIF-1 alpha), were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5V-flanking region isolated by PCR contains the gene promoter of human CRLR. Of signifi-
cance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5V-flanking region abolished such a response [27]. However, the mechanism of gene expression and regulation of RAMPs superfamily is unknown. It needs further consideration and investigation to clarify whether the factors that induce ADM gene overexpression also stimulate CRLR, RAMP2, RAMP3 gene expression and/or over-expressed ADM promotes directly its receptor system components’ gene expressions in a positive feedback manner. The concept has been evidenced by growing experimental data that endogenous ADM is an especially important paracrine/autocrine factor in cardiovascular homeostasis [15,28,29]. Administration of exogenous ADM or transfer of ADM gene improved obviously experimental hypertension, myocardial infarction and heart failure [28]. Furthermore, in mice in which the entire ADM gene was deleted, the homozygous knockout proved lethal at the embryonic stage, which indicates the importance of ADM in development of cardiovascular system [30,31]. The increase in ADM level was widely considered to have important compensatory and protective significance in cardiovascular diseases [15,29]. But the role of ADM receptor system components in regulating cardiovascular function is not well known. Resent works found that CRLR, RAMP2 and RAMP3 gene expressions were up-regulated in rats with hypertension, ischemic heart failure, obstructive nephropathy, septic shock, etc. [32 –34]. ADM could play a beneficial role in calcification-induced vascular damage, such as vasodilatation, protection of endothelium, attenuation of platelet aggregation and adhesion, inhibition of VSMCs proliferation, etc. Our previous literatures showed that administration of ADM inhibited obviously pathogenesis of vascular calcification in vivo and in vitro [16,17]. Therefore, we supposed that the high expression of ADM and its receptor system during vascular calcification might be an adaptive reaction with compensatory significance. 4.1. Summary The present study demonstrates that ADM and its receptor system components—CRLR, RAMP2 and RAMP3 were up-regulated in calcified VSMCs, which may be an adaptive reaction to inhibit the development of vascular calcification, and may be as a new target of precaution and treatment for the diseases relevant to vascular calcification.
Acknowledgements This work was supported by the State Major Basic Research Development Program (G2000056905) and Peking University Major Cardiovascular Research Program, a special fund for promotion of education. Ministry of Education PRC (985 Project).
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[18] Hay DL, Smith DM. Adrenomedullin receptors: molecular identity and function. Peptides 2001;22:1753 – 63. [19] Sexton PM, Albisto A, Morfis M, Tilakaratne N. Receptor activity modifying proteins. Cell Signal 2001;13:73 – 83. [20] Nagae T, Mukoyama M, Sugawara A, Mori K, Yahata K, Kasahara M, et al. Rat receptor activity modifying proteins (RAMPs) for adrenomedullin/CGRP receptor: cloning and upregulation in obstructive nephropathy. Biochem Biophys Res Commun 2000;270:89 – 93. [21] Qi YF, Bu DF, Niu DD, Shi YR, Wang SH, Pang YZ, Tang CS, Du JB. Effects of different peptide fragments derived from proadrenomedullin on gene expression of adrenomedullin gene expression. Peptides 2002;23:1141 – 7. [22] Balica M, Bostrom K, Shin V, Tillisch K, Demer LL. Calcification subpopulation of bovine aortic smooth muscle cells is responsive to 17h-estradiol. Circulation 1997;95:1954 – 60. [23] Lewis LK, Smith MW, Yandle TG, Tichards AM, Nicholls MG. Adrenomedullin(1 – 52) measured in human plasma by radioimmunoassay: plasma concentration, adsorption, and storage. Clin Chem 1998;44(3):571 – 7. [24] Tsuruda T, Kato J, Kitamura K, Kuwasako K, Imamura T, Koiwaya Y. Secretion of proadrenomedullin N-terminal 20 peptide from cultured neonatal rat cardiac cells. Life Sci 2001;69:239 – 45. [25] Hayashi M, Shimosawa T, Fujita T. Hyperglycemia increases vascular adrenomedullin expression. Biochem Biophys Res Commun 1999; 258:453 – 6. [26] McLatchie LM, Fraser NJ, Main MJ, Wise A, Brown J, Thompson N, et al. RAMPs regulate the transport and ligand specifity of the calcitonin receptor-like receptor. Nature 1998;393:333 – 9. [27] Nikitenko LL, Smith DM, Bicknell R, Rees MC. Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia. FASEB J 2003;17(11):1499 – 501. [28] Dobrzynski E, Wang C, Chao J, Chao L. Adrenomedullin gene delivery attenuates hypertension, cardiac remodeling, and renal injury in deoxycorticosterone acetate-salt hypertensive rats. Hypertension 2000; 36(6):995 – 1001. [29] Shimosawa T, Shibagaki Y, Ishibashi K, Kitamura K, Kangawa K, Kato S, et al. Adrenomedullin, an endogenous peptide, counteracts cardiovascular damage. Circulation 2002;105:106 – 11. [30] Hay DL, Smith DM. Knockout and transgenics confirm the importance of adrenomedullin in the vasculature. Trends Phamacol Sci 2001;22(2):57 – 9. [31] Shindo T, Kurihara Y, Nishimatsu H, Moriyama N, Kakoki M, Wang Y, et al. Vascular abnormalities and elevated blood pressure in mice lacking adrenomedullin gene. Circulation 2001;104:1964 – 71. [32] Oie E, Vinge LE, Yndestasd A, Sandberg C, Grogaaard HK, Attramadal H. Induction of a myocardial adrenomedullin signaling system during ischemic heart failure in rats. Circulation 2000;101:415 – 22. [33] Ono Y, Okano I, Kojima M, Okada K, Kangawa K. Decreased gene expression of adrenomedullin receptor in mouse lungs during sepsis. Biochem Biophys Res Commun 2002;71:197 – 202. [34] Totsune K, Takahashi K, Mackenzie HS, Murakami O, Arihara Z, Sone M, et al. Increased gene expression of adrenomedullin and adrenomedullin-receptor complexes receptor-activity modifying protein (RAMP)2 and calcitonin-receptor-like receptor (CRLR) in the hearts of rats with congestive heart failure. Clin Sci 1999;99:541 – 6.
Alterations of adrenomedullin and its receptor system components in calcified vascular smooth muscle cells Chun Shui Pan a, Yong Fen Qi a,b,c,*, Shu Heng Wang b, Jing Zhao b, Ding Fang Bu a, Gui Zhong Li a, Chao Shu Tang a,c b
a Institute of Cardiovascular Diseases, Peking University First Hospital, Beijing 100034, China Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100083, China c Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100083, China
Received 7 November 2003; received in revised form 18 February 2004; accepted 25 February 2004
Abstract Vascular calcification is a common finding in many cardiovascular diseases. Paracrine/autocrine changes in calcified vessels, and the secreted factors participate in and play an important role in the progress of calcification. Adrenomedullin (ADM) is a potent vasodilator peptide secreted by vascular smooth muscle cells (VSMCs) and vascular endothelial cells. Recently, receptor activity-modifying proteins (RAMPs) have been shown to transport calcitonin receptor-like receptor (CRLR) to the cell surface to present either as CGRP receptor or ADM receptor. In this work, we explored the production of ADM, alterations and significance of ADM mRNA and its receptor system components—CRLR and RAMPs mRNA in calcified VSMCs. Our results showed that calcium content, 45Ca2 + uptake and alkaline phosphatases (ALPs) activity in calcified VSMCs were increased, respectively, compared with control VSMCs. Content of ADM in medium was increased by 99% ( p < 0.01). Furthermore, it was found that the levels of ADM, CRLR, RAMP2 and RAMP3 mRNA in calcified cells were elevated, respectively, compared with that of control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA were significant correlation with ADM mRNA (r = 0.83, 0.92 and 0.93, respectively, all p’s < 0.01) in calcified VSMCs. The results show that calcified VSMCs generate an increased amount of ADM, up-regulate gene expressions of ADM and its receptor system components—CRLR, RAMP2 and RAMP3, suggesting an important role of ADM and its receptor system in the regulation of vascular calcification. D 2004 Elsevier B.V. All rights reserved. Keywords: Calcification; Vascular smooth muscle cells (VSMCs); ADM; CRLR; RAMPs
1. Introduction Vascular calcification is a common finding in many cardiovascular diseases, such as hypertension, atherosclerosis, diabetes, chronic renal failure, aging, aortic stenosis and prosthetic valve replacements [1 –3]. Calcified vessel causes a decreased function of vasodilatation, increased stiffness and promoted form of thrombus and atherosclerotic plaques rupture. Vascular calcification was believed to be an important risk factor of cardiovascular diseases [4,5]. However,
* Corresponding author. Department of Physiology and Pathophysiology, Institute of Cardiovascular Diseases, Peking University Health Science Center, Beijing 100083, China. Tel.: +86-10-82802183; fax: +86-1066556255. E-mail address: [email protected] (Y.F. Qi). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.02.020
the mechanism by which vascular calcification causes vascular dysfunction and remodeling is not clear yet [6,7]. Previously, calcification was generally considered as a passive calcium deposition in extracellular matrix and cells. The view, however, has changed in recent years, and the vascular calcification is considered as an active, regulative process similar to the osteogenesis [8]. During vascular calcification, the various vascular cells, including vascular smooth muscle cells (VSMCs), pericytes, fibroblasts and macrophages, transformed into osteoblast-like phenotype, which was characterized by an increase in alkaline phosphatase (ALP) activity, matrix vesicle formation and overexpression of bone morphogenetic proteins (BMPs) including BMP-2 and bone matrix proteins such as osteopontin, osteonectin, osteocalcin, etc. [5,9 – 11]. However, the changes in function of the vascular cells with altered phenotype and its pathophysiological significance remain
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unclear. It is common knowledge that paracrine/autocrine factors secreted from vascular cells contribute to circulatory homeostasis, and mediate pathogenesis of cardiovascular diseases. We supposed that phenotype-altered vascular cells not only had some properties of ossification, but also changed their paracrine/autocrine functions, and that the paracrine/autocrine dysfunction of calcified vascular vessels might play an important role in calcification-induced vascular damage. Adrenomedullin (ADM) was initially isolated from human pheochromocytoma cells by Kitamura et al. [12] in 1993 as a peptide capable of a potent and long-lasting hypotensive effect. ADM in blood circulation is mainly synthesized and secreted from vascular endothelial and smooth muscle cells. ADM has been shown to have vasodilatory, hypotensive and growth regulating properties, and is considered as an important regulatory peptide contributed to vascular homeostasis [13,14]. It was wellknown that ADM levels in plasma and cardiovascular tissues were compensatively elevated during cardiovascular diseases, as hypertension, heart failure, atherosclerosis, etc. [13,15]. Our previous researches found that ADM production was increased, and ADM gene was upregulated in calcified VSMCs in vitro and in calcified vessels of rat in vivo. Administration of ADM attenuated significantly pathogenesis of vascular calcification induced by vitamin D and nicotine [16,17]. However, it is unclear yet whether ADM receptor in calcified vessels changes or not, and which role do these changes play in pathogenesis of vascular calcification. It is well-known that VSMCs is highly responsive to ADM and rich in ADM receptors [18]. Recent researches showed that receptors bound with ADM were orphan L-1 and RDC-1 receptors [19]. In addition, another orphan receptor named calcitonin receptor-like receptor (CRLR) could bind with ADM when receptor activity-modifying protein (RAMP)2/RAMP3 existed on cellular membrane surface [20]. To facilitate the characteristics of alterations of autocrine and/or paracrine function in calcified vascular cells, we developed an in vitro calcification system in which diffused calcification could be induced by culturing rat VSMCs in the presence
of h-glycerophosphate. In this study, we observed the changes in ADM, and its receptor system components— CRLR and RAMP2/3 in calcified VSMCs to illustrate the role of ADM/ADM receptor pathway in pathogenesis of vascular calcification.
2. Materials and methods 2.1. Animals and reagents All animal experiments in this study were performed with the approval of the Animal Care Committee of the First Hospital, Peking University. Rat radioimmunoassay (RIA) kit for ADM was provided by Phoenix Pharmaceutical (St. Joseph, MO). h-Glycerophosphate and ALP assay kit were purchased from Sigma (St. Louis, MO). 45CaCl2 was obtained from NEN Life Sci Products (Boston, MA, USA) and Trizol was purchased from GIBICO. dNTP was purchased from Clontech. M-MuLV reverse transcriptase, Taq DNA polymerase, RNasin, Oligo(dT)15 Primer were provided by Promega. Other chemicals and reagents were of analytical grade. Sequences of oligonucleotide 10 primers for amplification are synthesized by SBS (China), as shown in Table 1. 2.2. Cell culture of VSMCs The explant culture method of VSMCs was described previously [21]. Briefly, rat thoracic aorta was cut into small pieces (about 2 – 3 mm each) after partial removal of external connective tissues, placed in DMEM medium supplemented with 4.5 g/l glucose, 10 mM sodium pyruvate and 20% fetal bovine serum and incubated at 37jC in an incubator containing 95% air and 5% CO2. VSMCs migrated from the explants were collected and maintained in growing medium (DMEM containing 10 mM sodium pyruvate and 15% fetal bovine serum). The a-actin examination of cultured cells confirmed a positive response. VSMCs of 5th to 8th passages were used for the experiments.
Table 1 Oligonucleotides used for the amplification Target ADM CRLR RAMP2 RAMP3 h-Actin
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Sequence
Size (bp)
Ann.T (jC)
Cycle
5V-CTCGACACTTCCTCGCAGTT-3V 5V-GCTGGAGCTGAGTGTGTCTG-3V 5V-CAACTGCTGGATCAGCTCAG-3V 5V-CATCGCTGATTGTTGACACC-3V 5V-TGAGGACAGCCTTCTGTC A-3V 5V-CATCGCCGTCTTTACTCC TC-3V 5V-CTTCTCCCTCTGTTGCTGCT-3V 5V-CACAGAAGCCGGTCAGTGT-3V 5V-ATCTGG AC CAC ACC TTC-3V 5V-AGCCAG GTC CAG ACG CA-3V
446
61
30
446
55
28
371
55
28
416
57
32
291
55
24
Expected sizes of the amplicons, annealing temperature (Ann.T) used for the PCR reaction.
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2.3. Cell calcification and assay of calcification ( 45Ca accumulation) [10] After confluence, the cells were inoculated on 24-well plates (104 cells/ml) in DMEM containing 10 mM sodium pyruvate supplemented with 15% FBS in the absence (i.e., growing medium, as control group) or in the presence of 10 mM h-glycerophosphate (i.e., calcification medium, as calcification group). The medium was replaced with fresh medium every 3 days. The treatment continued for 14 days. Twenty-four hours before the end of treatment, 1.0 ACi/ ml of 45CaCl2 was added in the medium. After 24 h of incubation, the medium was removed and the cell layer was washed five times with cold PBS, scraped into borosilicate tubes containing 0.5 ml of perchloric acid, and spun vigorously. Then, 0.5 ml of H2O2 was added and the suspensions were incubated for 60 min at 80 jC. After incubation, the mixture was dissolved in 1.0 ml of ethylene glycol monoethyl ether and spun vigorously, and radioactivity was measured by h-Liquid Scintillation Counting (Beckman, LS 6500). 2.4. Assay of cellular calcium content [10] After the end of incubation, the medium was removed and the cell layer was washed five times with cold PBS. Scraped cells were collected, and dissolved in HNO3 and diluted with a blank solution (27 nM KCl, 25 AM LaCl3), the calcium content was measured on an atomic absorption spectrophotometer at 422.7 nm. 2.5. Assay of AP activity [10] A cell-associated ALP activity assay was performed with a modification of the ALP assay kit from Sigma. The cells were rinsed three times with cold PBS and scraped into 200 Al of lysis buffer (0.2% NP-40 in 1 mM MgCl2) with a rubber policeman and sonicated for 10 s. Next, 1 ml of reaction mixture was added to each well. Reaction mixture was 221 alkaline buffer: stock substrate solution 1:1. Stock substrate solution was prepared by dissolving the contents of a 100-mg capsule of Sigma 104-phosphatase substrate in 25 ml of ddH2O. This mixture was then incubated for 30 min at 37 jC. The yellow color was indicative of ALP activity. The reaction was stopped by the addition of 12 Al of 1 M NaOH to each well, and absorbance was determined at 405 nm on spectrophotometer. ALP activity was calculated using U-nitrophenol as a standard, according to the kit’s instruction (Sigma). One unit was defined as the activity producing l nmol of U-nitrophenol for 30 min. 2.6. von Kossa calcium staining [22] Cultured cell monolayers were fixed in 0.1% glutaraldehyde in PBS for 30 min at room temperature. Cells were
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then washed twice with double-distilled water and incubated with 5% silver nitrate for 30 min at room temperature in the dark. The cells were washed gently with double-distilled water, exposed to ultraviolet light for 30 min, and counterstained with 0.1% eosin for 30 s. Calcium mineral stained black. Calcified nodules in each well were countered twice by use of phase-contrast microscope by two different individuals blinded to treatment conditions. 2.7. Radioimmunoassay for ADM [23] One milliliter of the medium at the end of cell culture was collected and immediately acidified with acetic acid to a final concentration of 1.0 M. The acidified medium was heated at 100 jC for 10 min to inactivate proteases, and applied to a Sep-Pak C18 cartridge (Millipore-Waters, Milford, MA, USA). After washing the cartridge with 10% CH3CN in 0.1% trifluoroacetic acid, the absorbed materials were eluted with 50% CH3CN in 0.1% trifluoroacetic acid, lyophilized and stored at 30 jC. The immunoreactive-ADM (ir-ADM) in the medium extract was measured using a specific RIA kit for ADM. Rabbit polyclonal anti-ADM was used for RIA in this kit. The IC50 of rat ADM was 13– 47 pg per tube, and the crossreactivity with rat ADM was 100%, and was zero with proadrenomedullin N-terminal 20 peptide, amylin and endothelin. Secretion of ADM from VSMCs was presented as fmol/105 cells. 2.8. Characterization of secreted ADM [24] To examine the molecular forms of ir-ADM, the extract of conditioned medium was analyzed by reverse-phase high-performance liquid chromatography (HPLC) with a column of TSK ODS 120 A (4.6 mm 25 cm, Tosoh, Tokyo, Japan). The extracts were eluted from the column with a linear gradient of 0 – 60% acetonitrile in 0.1% trifluoroacetic acid, and the ir-ADM in each fraction was measured with the RIA. The recovery of ir-ADM in the HPLC was greater than 80%. 2.9. Measurement of ADM, CRLR, RAMP2 and RAMP3 mRNA Total RNA of VSMCs was prepared by in situ lysis of the cells using Trizol (Gibco-BRL, Rockville, MD) reagent after washing the cells with ice-cold phosphate buffer solution. One microgram of total RNA was reverse-transcribed into single strand cDNA using M-MuLV reverse transcriptase and oligo(dT)15 primer. PCR was performed in a 0.2-ml tube containing VSMC cDNA 2 Al, 5 AM per each ADM-S and ADM-A primer mixture 1 Al, 2.5 mM per each dNTPs mixture 1 Al, 1.5 mM MgCl2 1.5 Al, 10 PCR buffer 2.5 Al and 1.25 unit Taq DNA polymerase, in a total volume of 25 Al. After denaturing at 95 jC for 5 min, PCR was run at 94 jC 30 s, 61 jC 30 s and 72
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2.10. Statistical analysis The values of various parameters were averaged from six independent experiments with duplication. Results of some experiments were normalized to total protein determined by Bradford’s method. The data were expressed as means F S.D. Comparisons across two groups were made using Student’s t test. Linear regression analysis was used to assess the correlation between variables. A p value < 0.05 was considered statistically significant.
3. Results 3.1. The general characteristics of VSMCs calcification Calcium mineral deposits formed by cultured VSMCs were assessed by the silver nitrate method (von Kossa staining). In calcified VSMCs, formation of multicellular nodules was shown. von Kossa staining for calcification, showed positive staining as black/brown areas within the main, large, nodular structures as shown in extracellular matrix and cytoplasma (Fig. 1). The content of calcium, 45 Ca2 + uptake and ALP activity in calcified VSMCs were increased by 118%, 174% and sevenfold (all p’s < 0.01), respectively, compared with that of control. (Table 2). 3.2. Increase in ADM secreted by calcified VSMCs and characteristic of secreted ADM
Fig. 1. (A) Calcification of multicellular nodules. von Kossa staining for calcification, showing positive staining as black/brown areas within the main, large, nodular structure (original magnification 40 ). (B) Formation of multicellular nodules in culture phase contrast microscopy showing rat VSMCs (original magnification 20 ).
jC 40 s for 30 cycles, 72 jC 5 min. Six microliters of PCR product was separated in a 1.5% agarose gel, and stained with ethidium bromide. The optical density of the 446-bp band was measured using the Gel Documentation System (Bio-Rad, Hercules, CA). Amplification of ADM cDNA was confirmed by digestion of the PCR products with restriction enzyme BglII. To calibrate the sample loaded in quantitative PCR, h-actin cDNA was determined at the same time. Two microliters of PCR product was amplified again using the two rat h-actin primers at 94 jC 30 s, 55 jC 30 s, and 72 jC 40 s for 24 cycles, 72 jC 5 min, and the optical density of the h-actin band (291 bp) was measured. The ratio of ADM mRNA/h-actin mRNA is the relative amount of ADM mRNA. The relative amount of CRLR, RAMP2, RAMP3 mRNA was determined according to the above method.
Content of ADM in calcified VSMCs medium was obviously increased by 99% as compared with control (17.82 F 1.68 versus 8.97 F 0.61 fmol/ml, p < 0.01). Molecular forms of ADM secreted into the medium were characterized by reverse-phase HPLC (Fig. 2). The irADM was composed of one major and one minor peak, and the major peak appeared at an elution position identical to that of synthetic rat ADM (1 – 50)-NH2, a whole active molecule of the rat ADM peptide. 3.3. Up-regulation of ADM mRNA in calcified VSMCs Semi-quantitative RT-PCR was performed to determine the ADM mRNA level of VSMCs. ADM gene products were identified by restriction enzyme BglII, and it was cut into two fragments, a 156-bp fragment and a 290-bp fragment confirmed that the ADM RT-PCR product was Table 2 General index of calcified VSMCs (n = 6, X F S.D.)
Control Calcified VSMC
45 Ca uptake (103 cpm/mg Pr)
Ca content (nmol/mg Pr)
ALP activity (U/mg Pr)
32.2 F 5.20 88.2 F 10.21**
46.8 F 7.23 102.2 F 18.23**
74 F 11 624 F 84**
ALP: alkaline phosphatase. ** p < 0.01 vs. control.
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Fig. 2. Analysis of reverse-phase HPLC of immunoreactive ADM (irADM) secreted from VSMCs into the media. A linear gradient of acetonitrile of 10 – 60% trifluoroacetic acid for 60 min at a flow rate of 1.0 ml/min. The arrow indicates the elution position of synthetic rat ADM (1 – 50)-NH2.
correct. The ADM mRNA level in calcified VSMCs was elevated by 78% ( p < 0.01) compared to control group. Results are shown in Fig. 3. 3.4. Up-regulation of ADM receptor system components mRNA in calcified VSMCs According to the methods described above we obtained the RT-PCR products of the CRLR, RAMP2 and RAMP3. These products were proved by the sequence analysis using the respectively relevant primer, which testified the RT-PCR products of CRLR, RAMP2 and RAMP3 were correct. The relative amounts of CRLR, RAMP2 and RAMP3 mRNA in calcified group were increased by 93.7%, 91.8% and 109.5% (all p’s < 0.01), respectively, compared with control. The elevated levels of CRLR, RAMP2 and RAMP3 mRNA
Fig. 4. Alterations of CRLR, RAMP2, RAMP3 mRNA in calcified VSMCs mRNA expression were determined by semi-quantitative RT-PCR analysis. CRLR: calcitonin receptor like receptor. RAMP: receptor activitymodifying protein. (A) RT-PCR products of CRLR, RAMP2 and RAMP3 mRNA were separated in a 1.5% agarose gel. (B) Quantitative analysis of changes in CRLR, RAMP2 and RAMP3 mRNA expression. Bars represent mean F S.D. of three individual samples. **p < 0.01, compared with control.
were in significant correlation with ADM mRNA (r = 0.83, 0.92 and 0.93, respectively, all p’s < 0.01) in calcified VSMCs. Results are shown in Fig. 4.
4. Discussion
Fig. 3. Change of ADM mRNA in VSMCs of rats. mRNA expressions were determined by semi-quantitative RT-PCR analysis. ADM: adrenomedullin. (A) RT-PCR products of ADM mRNA were separated in a 1.5% agarose gel. (B) Quantitative analysis of changes in ADM mRNA expression. Bars represent mean F S.D. of three individual samples. **p < 0.01, compared with control.
Vascular calcification is an active regulatory progress similar to osteogenesis and osteoporosis. Calcified vascular cells with osteoblast-like cell phenotype traits are cellular basis of vascular calcification pathogenesis [8]. The VSMCs transformed to osteoblast-like cells may not only result in an increase of vascular rigidity, which correlates with calcium phosphate deposition, but also influence vascular autocrine and/or paracrine function, and change vascular responsiveness to vasoactive substances [6,7]. In this study, we used cultured rat VSMCs treated with hglycerophosphate as a model of vascular calcification. hGlycerophosphate is a substrate for ALP. It can release inorganic phosphate to raise the local concentration of phosphorous, accelerate calcium and phosphorous deposition on cells and tissues, and induce formation of calcified nodule [10]. In the calcified VSMCs, we found that the calcium content, 45Ca2 + uptake and ALP activity were significantly increased compared with normal VSMCs. The alterations of these parameters demonstrated that calcified VSMCs had osteoblastic-like characteristics, which were in accordance with previous research [10]. It is known that VSMCs produce and secrete ADM, but cannot store it.
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The ADM level in the medium was generally considered as the amount of ADM synthesized by VSMCs [23]. In this work, we found that content of ADM in the medium was higher in calcified VSMCs than that of control VSMCs, and the level of ADM mRNA was also higher in calcified VSMCs than that of controls. The data show that the increase of ADM production in calcified VSMCs results from up-regulation of ADM gene expression. The ADM gene, located on chromosome 11, consists of four exons and three introns, and contains upstream transcription factor activator protein 2 (AP-2) and cAMP-regulated enhancer sequences [25]. Previous data showed that induction of AP2, secondary to activation of phospholipase C and protein kinase C, might be the mechanism by which growth factors such as fibroblast growth factor and platelet- and epidermalderived growth factors stimulated a high transcription and expression of the ADM gene in VSMCs [14,25]. The mechanism of ADM gene over-expression in calcified VSMCs, however, needs further investigation. A recent report [26] showed that CRLR could function as a calcitonin gene-related peptide (CGRP) receptor when coexpressed with a single transmembrane domain protein known as RAMP1, and as an ADM receptor when coexpressed with either RAMP2 or RAMP3. Interestingly it appears that the different RAMPs influence glycosylation leading to a detectable size difference between CRLR species with CGRP or ADM receptor activity. RAMP2/3 has been shown to modify the posttranslational processing of the CRLR, leading to high-affinity ADM binding [19]. In the present experiment, the data showed that the gene expressions of CRLR, RAMP2 and RAMP3 were obviously increased in calcified VSMCs, and the alterations of those mRNA levels were positive correlation with that of ADM mRNA. It was reported that ADM, CRLR, RAMP2 and RAMP3 mRNA were upregulated in many cardiovascular diseases, such as hypertension, heart failure, artherosclerosis, etc., and that activated ADM/ADM receptor system might play a compensatory protective role in cardiovascular diseases. RAMP2 gene is located on chromosome 17 and is composed of four exons separated by small introns. The first exon encodes 5V UTR sequence, while exon 4 is well-conserved and encode all of the C-terminal and transmembrane domains plus the distal 64 amino acids of N-terminal [18,19]. RAMP3 gene is located on chromosome 7 and comprise at least three exons with a large intron (>17 kb) separating the first two exons and a small (>5 kb) intron separating exons 2 and 3. It was reported that the CRLR genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor and hypoxia-inducible factor-1 alpha (HIF-1 alpha), were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5V-flanking region isolated by PCR contains the gene promoter of human CRLR. Of signifi-
cance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5V-flanking region abolished such a response [27]. However, the mechanism of gene expression and regulation of RAMPs superfamily is unknown. It needs further consideration and investigation to clarify whether the factors that induce ADM gene overexpression also stimulate CRLR, RAMP2, RAMP3 gene expression and/or over-expressed ADM promotes directly its receptor system components’ gene expressions in a positive feedback manner. The concept has been evidenced by growing experimental data that endogenous ADM is an especially important paracrine/autocrine factor in cardiovascular homeostasis [15,28,29]. Administration of exogenous ADM or transfer of ADM gene improved obviously experimental hypertension, myocardial infarction and heart failure [28]. Furthermore, in mice in which the entire ADM gene was deleted, the homozygous knockout proved lethal at the embryonic stage, which indicates the importance of ADM in development of cardiovascular system [30,31]. The increase in ADM level was widely considered to have important compensatory and protective significance in cardiovascular diseases [15,29]. But the role of ADM receptor system components in regulating cardiovascular function is not well known. Resent works found that CRLR, RAMP2 and RAMP3 gene expressions were up-regulated in rats with hypertension, ischemic heart failure, obstructive nephropathy, septic shock, etc. [32 –34]. ADM could play a beneficial role in calcification-induced vascular damage, such as vasodilatation, protection of endothelium, attenuation of platelet aggregation and adhesion, inhibition of VSMCs proliferation, etc. Our previous literatures showed that administration of ADM inhibited obviously pathogenesis of vascular calcification in vivo and in vitro [16,17]. Therefore, we supposed that the high expression of ADM and its receptor system during vascular calcification might be an adaptive reaction with compensatory significance. 4.1. Summary The present study demonstrates that ADM and its receptor system components—CRLR, RAMP2 and RAMP3 were up-regulated in calcified VSMCs, which may be an adaptive reaction to inhibit the development of vascular calcification, and may be as a new target of precaution and treatment for the diseases relevant to vascular calcification.
Acknowledgements This work was supported by the State Major Basic Research Development Program (G2000056905) and Peking University Major Cardiovascular Research Program, a special fund for promotion of education. Ministry of Education PRC (985 Project).
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