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Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts
Peptides 32 (2011) 2452–2458
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Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts Yong-Gang Zhang a,b,∗ , Ze-Jian Kuang a , Yan-Yan Mao a , Rui-Hong Wei c , Shi-Lin Bao a , Li-Biao Wu b , Yu-Guang Li a,∗∗ , Chao-Shu Tang d a
Department of Cardiovascular Diseases, First Affiliated Hospital, Shantou University Medical College, Shantou 515041, China Laboratory of Molecular Cardiology, First Affiliated Hospital, Shantou University Medical College, Shantou 515041, China c Department of Internal Medicine, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China d Institute of Cardiovascular Research, Peking University First Hospital, Beijing 100034, China b
a r t i c l e
i n f o
Article history: Received 13 November 2010 Received in revised form 15 October 2011 Accepted 16 October 2011 Available online 20 October 2011 Keywords: Osteopontin Urotensin II Migration Adventitial fibroblasts Signal transduction Peptide
a b s t r a c t Recent studies suggest that both osteopontin and urotensin II (UII) play critical roles in vascular remodeling. We previously showed that UII could stimulate the migration of aortic adventitial fibroblasts. In this study, we examined whether osteopontin is involved in UII-induced migration of rat aortic adventitial fibroblasts and examined the effects and mechanisms of UII on osteopontin expression in adventitial fibroblasts. Migration of adventitial fibroblasts induced by UII could be inhibited significantly by osteopontin antisense oligonucleotide (P < 0.01) but not sense or mismatch oligonucleotides (P > 0.05). Moreover, UII dose- and time-dependently promoted osteopontin mRNA expression and protein secretion in the cells, with maximal effect at 10−8 mol/l at 3 h for mRNA expression or at 12 h for protein secretion (both P < 0.01). Furthermore, the UII effects were significantly inhibited by its receptor antagonist SB710411 (10−6 mol/l), and Ca2+ channel blocker nicardipine (10−5 mol/l), protein kinase C (PKC) inhibitor H7 (10−5 mol/l), calcineurin inhibitor cyclosporine A (10−5 mol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10−5 mol/l) and Rho kinase inhibitor Y-27632 (10−5 mol/l). Thus, osteopontin is involved in the UII-induced migration of adventitial fibroblasts, and UII could upregulate osteopontin gene expression and protein synthesis in rat aortic adventitial fibroblasts by activating its receptor and the Ca2+ channel, PKC, calcineurin, MAPK and Rho kinase signal transduction pathways. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Urotensin II (UII) is the most potent vasoconstrictor in mammals [2]. Its vasoconstrictive potency is greater than that of endothelin1 (ET-1) [2]. Both UII and its receptor GPR 14 (UT receptor, UT) are expressed in many tissues, including spinal cord, certain brain areas [4], heart [2], endothelium, vascular smooth muscle cells (VSMCs) [2,11], adventitial fibroblasts [18,41], and renal tissues [30]. However, the cardiovascular system is the major target of UII
Abbreviations: Ang II, angiotensin II; CSA, cyclosporine A; DMEM, Dulbecco’s modified Eagle’s medium; ET-1, endothelin-1; MAPK, mitogen-activated protein kinase; OPN, osteopontin; PBS, phosphate-buffered saline; PKC, protein kinase C; TGF-1, transforming growth factor-1; UII, urotensin II; UT, UT receptor (GPR14); VSMCs, vascular smooth muscle cells. ∗ Corresponding author at: 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. ∗∗ Corresponding author. Tel.: +86 754 889 05337; fax: +86 754 882 59850. E-mail addresses: [email protected] (Y.-G. Zhang), [email protected] (Y.-G. Li). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.10.018
[2,4,11,41]. In addition to its role in vasoconstriction, UII exhibits mitogenic activity, which may stimulate proliferation of VSMCs [12], cardiac fibroblasts [33], and adventitial fibroblasts [39]. UII can also promote cardiac hypertrophy [9,38], foam cell formation [26], and myocardial fibrosis [40]; induce fibronectin and collagen production [33]; and stimulate secretion of other vasoactive factors, including ET-1 [32] and transforming growth factor 1 (TGF-1) [5]. Recently, UII was found to induce the migration of VSMCs [22] and adventitial fibroblasts [41]. The upregulation of UII and UT were reported in several cardiovascular diseases, including atherosclerosis [2,34], congestive heart failure, coronary heart disease [11], essential hypertension [28], and pulmonary hypertension [6], as well as renal failure, diabetes [10] and portal hypertension. UII was upregulated after balloon angioplasty, and treatment with SB-611812, the UT-specific antagonist, significantly reduced the ratio of intima to media area [24]. In addition, exogenous UII treatment significantly increased the intimal area, collagen content and cell proliferation of injured aortas and exacerbated the intimal hyperplasia [37]. These studies suggest an important role for UII in the pathogenesis of cardiovascular remodeling.
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Increasing evidence has suggested that differentiation of adventitial fibroblasts to adventitial myofibroblasts plays an important role in vascular remodeling [25,31]. Adventitial fibroblasts were found to migrate and might contribute to the formation of neointima after balloon injury [27]. The migration of adventitial fibroblasts is one of the common pathological processes in vascular remodeling [35]. Factors involved in the process include angiotensin II (Ang II) [31] and TGF- [8]. Recently, we found that UII could stimulate migration and phenotypic differentiation of adventitial fibroblasts mainly in an autocrine/paracrine manner [39]. However, the significance and mechanism of the effect of UII on adventitial fibroblast migration have not been completely clarified. Studies have identified that osteopontin (OPN) is an important factor involved in cell migration and vascular remodeling. OPN is a matricellular protein present in bodily fluids and tissues. Through interactions with integrins, OPN mediates cell migration, adhesion, and survival in many cell types [21]. Recently, OPN was found to be a predictor of coronary artery disease [1]. In vitro studies have demonstrated that OPN promotes migration and proliferation of VSMCs and accumulation of extracellular matrices [17]. Many mediators, such as tumor necrosis factor ␣ (TNF-␣), interleukin 1, Ang II, TGF-, and hyperglycemia strongly induce OPN expression [20]. Recently, OPN was found to be an important stimulator of adventitial fibroblasts [29]. UII could induce the migration and phenotypic conversion of adventitial fibroblasts; however, the crosstalk between UII and OPN in activation of adventitial fibroblasts is unknown. Therefore, in the present study, we explored whether OPN is involved in the migration of adventitial fibroblasts induced by UII and examined the effects of UII on OPN mRNA and protein production in rat adventitial fibroblasts. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 180–200 g were supplied by the Animal Center, Shantou University Medical College. Animal care and experimental protocols were in compliance with the Animal Management Rules of China (Documentation No. 545, 2001, Ministry of Science and Technology, China) and the Guide for Care and Use of Laboratory Animals, Shantou University Medical College. 2.2. Materials and instruments Rat UII (pEHGTAPECFWKYCI) and SB710411 were from Phoenix Pharmaceuticals (Belmont, CA, USA). Fetal bovine serum was from Hyclone (Logan, UT, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from GiBco (Invitrogen-Gibco, Carlsbad, CA, USA); Y-27632, H7, cyclosporin A (CSA) and nicardipine were from Sigma (St. Louis, MO, USA). PD98059 was from Calbiochem (Darmstadt, Germany). The rat OPN ELISA kit was from Immuno-Biological Laboratories (Gunma, Japan). Oligonucleotides were synthesized by Sangon (Shanghai, China). The sequences of oligonucleotide primers were OPN sense 5 -ACAGTATCCCGATGCCACA-3 , antisense 5 -GTTTCCACGCTTGGTTCAT-3 , -actin sense 5 -ATCTGGCACCACACCTTC-3 , and antisense 5 -AGCCAGGTCCAGACGCA-3 , for calibration of sample loading. Three phosphorothioate oligonucleotides of OPN were synthesized by Sangon (Shanghai, China). We used the sequences for antisense oligonucleotides, 5 -ACCACTGCCAGTCTCAT-3 ; sense oligonucleotides, 5 -ATGAGACTGGCAGTGGTT-3 ; and mismatch oligonucleotides, 5 -AACTACTATCAGTCTCGT-3 . Other chemicals and reagents were of analytical grade.
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2.3. Cell culture Adventitial fibroblasts from aortas were prepared as described [15] with modification. Briefly, rats were anesthetized with isoflurane and rapidly decapitated. The full length of the thoraco-abdominal aorta was isolated under a pool of 4 ◦ C phosphate-buffered saline (PBS) after gently opening the parietal pleura, separating 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 the use of two forceps. The remaining tissues, predominantly adventitia, were cut into small pieces and placed in 25-cm2 tissue culture flasks in DMEM containing 20% fetal bovine serum (FBS). The explants were cultivated until large colonies of cells formed surround the explants. Fibroblasts grew from these explants within 4–7 days. After achieving confluence, cells were harvested with trypsin and used for experiments at passage 3–5. The morphology and growth characteristics of the cells were typical of fibroblasts and were distinguished from smooth muscle cells by the absence of a “hill and valley” growth pattern and the lack of ␣-SM actin staining. To examine OPN mRNA expression of adventitial fibroblasts induced by UII, the cultured cells were incubated in DMEM with 10% fetal bovine serum (FBS), and then incubated with serum-free medium for 24 h. After synchronization of adventitial fibroblasts, the medium was changed to DMEM without serum. Thereafter, cells were incubated in the presence or absence of UII. To examine the molecular basis of the action of UII, we used the inhibitors for UT antagonist SB710411 (10−6 mol/l), Ca2+ channel blocker nicardipine (10−5 mol/l), PKC inhibitor H7 (10−5 mol/l), Rho protein kinase inhibitor Y-27632 (10−5 mol/l), calcineurin inhibitor CSA (10−5 mol/l) and MAPK inhibitor PD98059 (10−5 mol/l). Cells were divided into the following groups for treatment: (1) control: culture in serum-free DMEM and no UII; (2) UII: 10−10 , 10−9 , 10−8 or 10−7 mol/l UII added to the medium; (3) UII + inhibitors: pretreatment in DMEM for 0.5 h with SB710411 (10−6 mol/l), PD98059 (10−5 mol/l), CSA (10−5 mol/l), nicardipine (10−5 mol/l), H7 (10−5 mol/l), or Y-27632 (10−5 mol/l) then UII (10−8 mol/l). The cells and medium were collected after 1–24 h of incubation. 2.4. RNA isolation Total RNA was isolated directly from cells by use of Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, Trizol reagent was added directly to each dish with vigorous pipetting and then transferred to a 1.5-ml eppendorf tube. After incubation at 30 ◦ C for 5 min, 200 l of chloroform was added. The mixture was vortexed and centrifuged at 12,000 rpm for 15 min. RNA was precipitated with an equal volume of isopropanol and washed with 75% ethanol. Then, RNA was air-dried and resuspended in diethyl pyrocarbonate-treated water. The RNA preparation was treated with DNase I to remove residual traces of DNA, purified with the phenol–chloroform method and precipitated with ethanol. RNA concentration and purity were determined on a UV spectrophotometer (Bio-Rad, Hercules, CA, USA) with 260 nm absorbance and 260/280 nm absorbance ratio, respectively. Agarose gel electrophoresis (2%) and Goldview nucleic acid staining were used to examine RNA integrity. 2.5. RT-PCR Reverse transcription involved use of a commercially available kit according to the manufacturer’s instructions. The first strand-cDNA was synthesized by the Reverse Transcription System
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(Promega, Madison, USA). Briefly, 1 g of total RNA was placed in a microcentrifuge tube and incubated at 70 ◦ C for 10 min, then centrifuged briefly in a microcentrifuge, and placed on ice. The reaction was prepared by adding the following reagents (final concentration of reaction components): 5 mmol/l MgCl2 , 1× reverse transcription buffer, 1 mmol/l each dNTP mixture, 1 U/l recombinant RNasin® ribonuclease inhibitor, 15 U/g AMV reverse transcriptase, 0.5 g Oligo(dT)15 primer and nuclease-free water in a total volume of 20 l, then co-incubating according to the manufacturer’s protocol. PCR involved use of a PCR instrument (Bio-Rad, Hercules, CA, USA) with GoTaq® Green Master Mix (Promega, Madison, USA). Briefly, 1 l cDNA mixture underwent amplification in a 25 l mixture containing 12.5 l GoTaq® Green Master Mix, 11 l nuclease-free water and 0.1 mol/l each primer set. PCR conditions were initial denaturation at 95 ◦ C for 2 min, then 30 cycles of denaturation at 95 ◦ C for 0.5 min, annealing for 0.5 min at 55 ◦ C, extension at 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 5 min. As an internal control for each PCR reaction, -actin cDNA was amplified for each sample under the same conditions. The PCR products were separated on 2% agarose gels and quantified with use of Quantity One 4.4.0 software (Bio-Rad, Hercules, CA, USA). Results were expressed as the ratio of OPN PCR product (174 bp) to -actin PCR product (291 bp) for each sample. All experiments were repeated 6 times.
2.6. Enzyme-linked immunosorbent assay (ELISA) OPN secretion from adventitial fibroblasts was measured by ELISA according to the manufacturer’s instructions. After incubation, the culture medium was collected, centrifuged immediately, and added to 96-well plastic plates. Absorbance of colored products was determined by use of a microplate reader set to 450 nm. Experiments were performed in duplicate and repeated 4 times.
Data analysis involved use of Prisms software. A two-tailed P value <0.05 was considered statistically significant. 3. Results 3.1. The role of OPN antisense oligonucleotide in the migration of adventitial fibroblasts induced by UII UII significantly enhanced the migration of adventitial fibroblasts (P < 0.01) as compared with control treatment (Fig. 1). The effect of UII was significantly inhibited by OPN antisense oligonucleotide (P < 0.01) but not sense or mismatch oligonucleotides as compared with UII treatment alone (P > 0.05). 3.2. The effect of UII on OPN mRNA expression in adventitial fibroblasts To validate that OPN was involved in UII actions, we examined the direct effect of UII on OPN mRNA expression. UII significantly stimulated OPN mRNA expression in adventitial fibroblasts in a time-dependent manner (Fig. 2). The effect of UII (10−8 mol/l) on OPN mRNA expression began to increase at 1 h and peaked at 3 h (all P < 0.01), then gradually decreased to the control level and lower from 12 to 24 h (P > 0.05). UII affected OPN mRNA expression in a concentration-dependent manner (Fig. 3). Maximal stimulation was reached at 10−8 mol/l UII (P < 0.01). Furthermore, the effect of UII on OPN mRNA expression was inhibited by the UT antagonist SB710411 (10−6 mol/l, P < 0.01), Rho protein kinase inhibitor Y-27632 (10−5 mol/l, P < 0.01), PKC inhibitor H7 (10−5 mol/l, P < 0.01), calcineurin inhibitor CSA (10−5 mol/l, P < 0.01), Ca2+ channel blocker nicardipine (10−5 mol/l, P < 0.01), and MAPK kinase inhibitor PD98059 (10−5 mol/l, P < 0.01) (Figs. 4 and 5), which suggests that these pathways might be involved in OPN mRNA expression induced by UII.
2.7. Cell migration assay We used antisense technology to investigate the role of OPN in migration of adventitial fibroblasts induced by UII by the transwell method (Corning), as described previously [29], which allows cells to migrate through a 8-m pore size polycarbonate membrane. Briefly, cultured adventitial fibroblasts were trypsinized, washed, and resuspended in serum-free DMEM (5 × 104 cells/ml). This suspension (500 l) was added to the lower transwell chamber so that they would be 90–95% confluent at the time of transfection. For each transfection sample, 1.0 g antisense (sense/mismatch) oligodeoxynucleotides (oligonucleotides) were diluted in 50 l DMEM, mixed with 50 l DMEM containing 3 l lipofectamine, and incubated at room temperature for 20 min. The mixture was then added to 500 l serum-free DMEM, and cells were incubated in this medium at 37 ◦ C. After 8 h, UII (10−8 mol/l) was then added to the medium for co-incubation for 24 h. Then 200 l cell suspension (1 × 105 cells/ml) was added to the upper transwell chamber. After incubation for 6 h at 37 ◦ C in the presence of 5% CO2 , the filters were removed, and cells remaining on the upper surface of the membrane were removed with a cotton swab. Then membranes were washed with PBS, and cells present beneath the membrane were fixed with 100% methanol for 2 min and stained with hematoxylin and eosin. The migration of cells was quantified by cell counts of 5 random fields at 200× magnification in each membrane. Each experiment was performed in triplicate.
2.8. Statistical analysis Results are shown as mean ± SE. Comparisons involved one-way ANOVA, followed by the Newman–Keuls multiple comparison test.
3.3. The effect of UII on OPN protein secretion from adventitial fibroblasts The effect of UII on OPN protein secretion from adventitial fibroblasts was evaluated by ELISA. UII stimulated OPN secretion in a time-dependent manner (Fig. 6). OPN secretion began to increase at 6 h and peaked at 12 h (all P < 0.05). The peak OPN protein secretion was at 10−8 mol/l UII (P < 0.01) (Fig. 7). This effect of UII on OPN protein secretion was inhibited by pretreatment with SB710411 (10−6 mol/l, P < 0.05), Y-27632, H7, CSA, nicardipine, and PD98059 (all 10−5 mol/l, all P < 0.05) (Figs. 8 and 9). 4. Discussion Recent studies suggest that both OPN and UII play critical roles in vascular remodeling [13]. In this study, we examined the role of OPN in UII-induced migration of adventitial fibroblasts and the effects of UII on OPN expression in the fibroblasts. Rat UII could induce migration of adventitial fibroblasts. This effect could be inhibited by OPN antisense but not sense or mismatch oligonucleotides, which indicates that OPN plays an important role in the migration induced by UII. Moreover, UII significantly increased both OPN mRNA and protein production in adventitial fibroblasts in a time- and concentration-dependent manner. The gene and protein expression were coincidental. Furthermore, SB710411, PD98059, nicardipine, H7, CSA and Y-27632, inhibitors or blockers of UT, MAPK, Ca2+ channel, PKC, calcineurin and Rho kinase, respectively, could inhibit the increase in OPN mRNA and protein expression induced by UII, so activation of these pathways might be involved in the UII-induced OPN biosynthesis.
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Fig. 1. Effects of antisense, sense, and mismatch oligonucleotides of OPN on adventitial fibroblast migration induced by UII. Cell migration was measured in transwell chambers. (A) Control; (B) UII; (C) UII + antisense oligonucleotides; (D) UII + sense oligonucleotides; (E) UII + mismatch oligonucleotides; (F) migrated cell numbers/field (n = 4 experiments). *P < 0.05, **P < 0.01 vs. control, ## P < 0.01 vs. UII.
Strong evidence indicates that UII is implicated in cardiovascular diseases. Specifically, the crosstalk between UII and other vascular regulators plays an important role in the pathophysiological process of the diseases, such as atherosclerosis [2,23], vascular remodeling and myocardial fibrosis [5,33,40]. Ang II and ET-1 were found to modulate UII-induced increase in myocardial distensibility [7], and UII could induce ET-1 expression in rat aortic SMCs via epidermal growth factor receptor transactivation [32]. As well, secretion of TGF-1 was upregulated in neonatal cardiac fibroblasts by UII, and TGF-1 was involved in UII-induced collagen synthesis in the cells [5]. UII also promoted TGF-1 production in rat
proximal tubular epithelial cells [30]. In the present study, we found that UII could induce the expression of OPN, an important factor in cell migration and vascular remodeling, and OPN antisense oligonucleotides could inhibit the effects of UII on migration of adventitial fibroblasts, so OPN is involved in the UII-induced migration of adventitial fibroblasts. As well, crosstalk exists
Fig. 2. UII time-dependently increased OPN mRNA expression in rat adventitial fibroblasts. Rat adventitial fibroblasts were incubated with UII (10−8 mol/l) for the times indicated. OPN mRNA expression was examined by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of ratio of OPN to -actin relative to the control group from 6 independent experiments. **P < 0.01 vs. control.
Fig. 3. UII dose-dependently increased OPN mRNA in rat adventitial fibroblasts. Rat adventitial fibroblasts were treated with UII (10−10 –10−7 mol/l) for 6 h. OPN mRNA expression was examined by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of ratio of OPN to -actin relative to the control group from 6 independent experiments (n = 6). *P < 0.05; **P < 0.01 vs. control.
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Fig. 4. UII promoted OPN mRNA expression in a UT-dependent way in rat adventitial fibroblasts. Cells were preincubated with SB710411 (SB), the UT antagonist, for 30 min, then with UII (10−8 mol/l) and the antagonist for 6 h. OPN mRNA expression was estimated by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of OPN to -actin relative to the control from 6 independent experiments. **P < 0.01 vs. control; ## P < 0.01 vs. UII.
Fig. 6. UII time-dependently increased OPN protein secretion from rat adventitial fibroblasts. Rat adventitial fibroblasts were treated with UII (10−8 mol/l) for the times indicated, the culture medium was collected, and protein level of OPN was determined by ELISA; optical density was read at 450 nm wavelength (n = 4 experiments). *P < 0.05 vs. control.
Fig. 7. UII dose-dependently increases OPN protein secretion from rat adventitial fibroblasts. Rat adventitial fibroblasts were treated with UII (10−10 –10−7 mol/l) for 12 h, the culture medium was collected, and OPN was determined by ELISA, with optical density read at 450 nm wavelength (n = 4). *P < 0.05, **P < 0.01 vs. control.
Fig. 5. Effect of different inhibitors on UII-induced OPN mRNA expression in rat adventitial fibroblasts. Cells were preincubated with Y-27632 (Y), H7, cyclosporine A (CSA), nicardipine (Ni), or PD98059 (PD; all 10−5 mol/l) for 30 min, then incubated with UII (10−8 mol/l) and the inhibitors for 6 h. OPN mRNA expression was examined by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of OPN to -actin relative to the control group from 6 independent experiments. **P < 0.01 vs. control; ## P < 0.01 vs. UII.
between UII and OPN in vascular regulation and vascular remodeling. The mechanisms of the UII effect on OPN expression are similar to the effects of UII on the induction of ET-1, TGF-1 and cell adhesion molecules. UII is able to stimulate TGF-1 secretion from neonatal cardiac fibroblasts and proximal tubular epithelial cells in rats via UT activation [5,30]. In cultured human coronary artery endothelial cells, UII increased the expression of tissue factor and cell adhesion molecules (VCAM-1/ICAM-1) through Rho kinase activation [3]. Moreover, the mechanisms involved in the UII-induced OPN expression are analogous to those of UII effects on cell migration. UII enhanced migration of human aortic SMCs through activation of an ERK-dependent pathway [22]. We previously suggested that UII may stimulate the migration of adventitial
Fig. 8. UII promotes OPN protein secretion in a UT-dependent way in rat adventitial fibroblasts. Rat adventitial fibroblasts were preincubated with UT antagonist SB710411 (SB; 10−6 mol/l) for 30 min, then incubated with UII (10−8 mol/l) and the antagonist for 12 h. The culture medium was then collected, and OPN was determined by ELISA, with optical density read at 450 nm wavelength (n = 4). *P < 0.05 vs. control, # P < 0.05 vs. UII.
fibroblasts through PKC, MAPK, calcineurin, and Rho kinase signal transduction pathways [41]. Recently, UII was found to induce migration of endothelial progenitor cells via activation of the RhoA/Rho kinase pathway [36]; UII could induce OPN expression via the UT, Ca2+ channel, PKC, MAPK, calcineurin and Rho kinase pathways, which provided new insights into UII actions. In addition, UII was found to induce adventitial fibroblast proliferation, collagen synthesis and secretion, and phenotypic differentiation through the activation of UT, PKC, MAPK, calcineurin, Rho protein kinase
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Fig. 9. Effects of different inhibitors on UII-induced OPN protein secretion in rat adventitial fibroblasts. Rat adventitial fibroblasts were preincubated with H7, cyclosporine A (CSA), Y-27632 (Y), PD98059 (PD), or nicardipine (Ni; all 10−5 mol/l) for 30 min, then incubated with UII (10−8 mol/l) and the inhibitors for 12 h. The culture medium was collected, and OPN was determined by ELISA; optical density was read at 450 nm wavelength (n = 4). **P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs. UII.
and/or calcium channel [39,41]. These signal pathways may play important roles in the activation of adventitial fibroblasts induced by UII. Vascular adventitial fibroblasts play a key role in vascular remolding through phenotypic conversion, proliferation and migration. Numerous cytokines and factors may influence these processes [19,25]. Both OPN and UII are involved in atherosclerotic plaque and neointima formation after angioplasty [2,13,37]. OPN also enhanced the migratory ability of cultured aortic adventitial fibroblasts from spontaneously hypertensive rats [29]. Fibroblast growth factor 1 could upregulate OPN expression via Src/MEK/MAP kinase signaling pathways in rat aortic SMCs, with further mediation of the migration of adventitial fibroblasts [16]. Recently, the autocrine expression of OPN was found to play a major role in SMC migration induced by platelet-derived growth factor [14]. Our previous study demonstrated that UII could induce phenotypic conversion and migration of vascular adventitial fibroblasts as an autocrine and paracrine factor [39,41]. The present study indicates that UII effects on vascular remodeling may partly involve the autocrine expression of OPN in the adventitial fibroblasts. In conclusion, we revealed that UII-induced migration of adventitial fibroblasts could be inhibited by OPN antisense oligonucleotides. In addition, UII significantly increased both the mRNA and protein expression of OPN in adventitial fibroblasts, via the activation of the UT, and the Ca2+ channel, MAPK, PKC, calcineurin and Rho kinase signal transduction pathways. This study provides new insights into UII actions that UII may act partly via the autocrine production of OPN in the adventitial fibroblasts. Simultaneous blockade of UII and of OPN expression could be a promising therapeutic strategy to limit vascular remodeling. Acknowledgments This project was supported by National Natural Science Foundation of China (No. 30971273) and the Natural Science Foundation of Guangdong Province (No. 9151051501000016) of China. The experiment was completed with the assistance from the Laboratory of Molecular Biology & Cardiology, First Affiliated Hospital, Shantou University Medical College. References [1] Abdel-Azeez HA, Al-Zaky M. Plasma osteopontin as a predictor of coronary artery disease: association with echocardiographic characteristics of atherosclerosis. J Clin Lab Anal 2010;24:201–6.
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[16] Li G, Oparil S, Kelpke SS, Chen YF, Thompson JA. Fibroblast growth factor receptor-1 signaling induces osteopontin expression and vascular smooth muscle cell-dependent adventitial fibroblast migration in vitro. Circulation 2002;106:854–9. [17] Li JJ, Han M, Wen JK, Li AY. Osteopontin stimulates vascular smooth muscle cell migration by inducing FAK phosphorylation and ILK dephosphorylation. Biochem Biophys Res Commun 2007;356:13–9. [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] Liu Y, Liang C, Liu X, Liao B, Pan X, Ren Y, et al. AGEs increased migration and inflammatory responses of adventitial fibroblasts via RAGE, MAPK and NF-kappaB pathways. Atherosclerosis 2010;208:34–42. [20] Lorenzen JM, Neunhoffer H, David S, Kielstein JT, Haller H, Fliser D. Angiotensin II receptor blocker and statins lower elevated levels of osteopontin in essential hypertension—results from the EUTOPIA trial. Atherosclerosis 2010;209:184–8. [21] Lund SA, Giachelli CM, Scatena M. The role of osteopontin in inflammatory processes. J Cell Commun Signal 2009;3:311–22. [22] Matsusaka S, Wakabayashi I. Enhancement of vascular smooth muscle cell migration by urotensin II. Naunyn Schmiedebergs Arch Pharmacol 2006;373:381–6. [23] Papadopoulos P, Bousette N, Al-Ramli W, You Z, Behm DJ, Ohlstein EH, et al. Targeted overexpression of the human urotensin receptor transgene in smooth muscle cells: effect of UT antagonism in ApoE knockout mice fed with Western diet. Atherosclerosis 2009;204:395–404. [24] 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. [25] 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. [26] Shiraishi Y, Watanabe T, Suguro T, Nagashima M, Kato R, Hongo S, et al. Chronic urotensin II infusion enhances macrophage foam cell formation and atherosclerosis in apolipoprotein E-knockout mice. J Hypertens 2008;26:1955–65. [27] Siow RC, Mallawaarachchi CM, Weissberg PL. Migration of adventitial myofibroblasts following vascular balloon injury: insights from in vivo gene transfer to rat carotid arteries. Cardiovasc Res 2003;59:212–21. [28] 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. [29] Sun AJ, Gao PJ, Liu JJ, Ji KD, Zhu DL. 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Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts Yong-Gang Zhang a,b,∗ , Ze-Jian Kuang a , Yan-Yan Mao a , Rui-Hong Wei c , Shi-Lin Bao a , Li-Biao Wu b , Yu-Guang Li a,∗∗ , Chao-Shu Tang d a
Department of Cardiovascular Diseases, First Affiliated Hospital, Shantou University Medical College, Shantou 515041, China Laboratory of Molecular Cardiology, First Affiliated Hospital, Shantou University Medical College, Shantou 515041, China c Department of Internal Medicine, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, China d Institute of Cardiovascular Research, Peking University First Hospital, Beijing 100034, China b
a r t i c l e
i n f o
Article history: Received 13 November 2010 Received in revised form 15 October 2011 Accepted 16 October 2011 Available online 20 October 2011 Keywords: Osteopontin Urotensin II Migration Adventitial fibroblasts Signal transduction Peptide
a b s t r a c t Recent studies suggest that both osteopontin and urotensin II (UII) play critical roles in vascular remodeling. We previously showed that UII could stimulate the migration of aortic adventitial fibroblasts. In this study, we examined whether osteopontin is involved in UII-induced migration of rat aortic adventitial fibroblasts and examined the effects and mechanisms of UII on osteopontin expression in adventitial fibroblasts. Migration of adventitial fibroblasts induced by UII could be inhibited significantly by osteopontin antisense oligonucleotide (P < 0.01) but not sense or mismatch oligonucleotides (P > 0.05). Moreover, UII dose- and time-dependently promoted osteopontin mRNA expression and protein secretion in the cells, with maximal effect at 10−8 mol/l at 3 h for mRNA expression or at 12 h for protein secretion (both P < 0.01). Furthermore, the UII effects were significantly inhibited by its receptor antagonist SB710411 (10−6 mol/l), and Ca2+ channel blocker nicardipine (10−5 mol/l), protein kinase C (PKC) inhibitor H7 (10−5 mol/l), calcineurin inhibitor cyclosporine A (10−5 mol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10−5 mol/l) and Rho kinase inhibitor Y-27632 (10−5 mol/l). Thus, osteopontin is involved in the UII-induced migration of adventitial fibroblasts, and UII could upregulate osteopontin gene expression and protein synthesis in rat aortic adventitial fibroblasts by activating its receptor and the Ca2+ channel, PKC, calcineurin, MAPK and Rho kinase signal transduction pathways. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Urotensin II (UII) is the most potent vasoconstrictor in mammals [2]. Its vasoconstrictive potency is greater than that of endothelin1 (ET-1) [2]. Both UII and its receptor GPR 14 (UT receptor, UT) are expressed in many tissues, including spinal cord, certain brain areas [4], heart [2], endothelium, vascular smooth muscle cells (VSMCs) [2,11], adventitial fibroblasts [18,41], and renal tissues [30]. However, the cardiovascular system is the major target of UII
Abbreviations: Ang II, angiotensin II; CSA, cyclosporine A; DMEM, Dulbecco’s modified Eagle’s medium; ET-1, endothelin-1; MAPK, mitogen-activated protein kinase; OPN, osteopontin; PBS, phosphate-buffered saline; PKC, protein kinase C; TGF-1, transforming growth factor-1; UII, urotensin II; UT, UT receptor (GPR14); VSMCs, vascular smooth muscle cells. ∗ Corresponding author at: 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. ∗∗ Corresponding author. Tel.: +86 754 889 05337; fax: +86 754 882 59850. E-mail addresses: [email protected] (Y.-G. Zhang), [email protected] (Y.-G. Li). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.10.018
[2,4,11,41]. In addition to its role in vasoconstriction, UII exhibits mitogenic activity, which may stimulate proliferation of VSMCs [12], cardiac fibroblasts [33], and adventitial fibroblasts [39]. UII can also promote cardiac hypertrophy [9,38], foam cell formation [26], and myocardial fibrosis [40]; induce fibronectin and collagen production [33]; and stimulate secretion of other vasoactive factors, including ET-1 [32] and transforming growth factor 1 (TGF-1) [5]. Recently, UII was found to induce the migration of VSMCs [22] and adventitial fibroblasts [41]. The upregulation of UII and UT were reported in several cardiovascular diseases, including atherosclerosis [2,34], congestive heart failure, coronary heart disease [11], essential hypertension [28], and pulmonary hypertension [6], as well as renal failure, diabetes [10] and portal hypertension. UII was upregulated after balloon angioplasty, and treatment with SB-611812, the UT-specific antagonist, significantly reduced the ratio of intima to media area [24]. In addition, exogenous UII treatment significantly increased the intimal area, collagen content and cell proliferation of injured aortas and exacerbated the intimal hyperplasia [37]. These studies suggest an important role for UII in the pathogenesis of cardiovascular remodeling.
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Increasing evidence has suggested that differentiation of adventitial fibroblasts to adventitial myofibroblasts plays an important role in vascular remodeling [25,31]. Adventitial fibroblasts were found to migrate and might contribute to the formation of neointima after balloon injury [27]. The migration of adventitial fibroblasts is one of the common pathological processes in vascular remodeling [35]. Factors involved in the process include angiotensin II (Ang II) [31] and TGF- [8]. Recently, we found that UII could stimulate migration and phenotypic differentiation of adventitial fibroblasts mainly in an autocrine/paracrine manner [39]. However, the significance and mechanism of the effect of UII on adventitial fibroblast migration have not been completely clarified. Studies have identified that osteopontin (OPN) is an important factor involved in cell migration and vascular remodeling. OPN is a matricellular protein present in bodily fluids and tissues. Through interactions with integrins, OPN mediates cell migration, adhesion, and survival in many cell types [21]. Recently, OPN was found to be a predictor of coronary artery disease [1]. In vitro studies have demonstrated that OPN promotes migration and proliferation of VSMCs and accumulation of extracellular matrices [17]. Many mediators, such as tumor necrosis factor ␣ (TNF-␣), interleukin 1, Ang II, TGF-, and hyperglycemia strongly induce OPN expression [20]. Recently, OPN was found to be an important stimulator of adventitial fibroblasts [29]. UII could induce the migration and phenotypic conversion of adventitial fibroblasts; however, the crosstalk between UII and OPN in activation of adventitial fibroblasts is unknown. Therefore, in the present study, we explored whether OPN is involved in the migration of adventitial fibroblasts induced by UII and examined the effects of UII on OPN mRNA and protein production in rat adventitial fibroblasts. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats weighing 180–200 g were supplied by the Animal Center, Shantou University Medical College. Animal care and experimental protocols were in compliance with the Animal Management Rules of China (Documentation No. 545, 2001, Ministry of Science and Technology, China) and the Guide for Care and Use of Laboratory Animals, Shantou University Medical College. 2.2. Materials and instruments Rat UII (pEHGTAPECFWKYCI) and SB710411 were from Phoenix Pharmaceuticals (Belmont, CA, USA). Fetal bovine serum was from Hyclone (Logan, UT, USA). Dulbecco’s modified Eagle’s medium (DMEM) was from GiBco (Invitrogen-Gibco, Carlsbad, CA, USA); Y-27632, H7, cyclosporin A (CSA) and nicardipine were from Sigma (St. Louis, MO, USA). PD98059 was from Calbiochem (Darmstadt, Germany). The rat OPN ELISA kit was from Immuno-Biological Laboratories (Gunma, Japan). Oligonucleotides were synthesized by Sangon (Shanghai, China). The sequences of oligonucleotide primers were OPN sense 5 -ACAGTATCCCGATGCCACA-3 , antisense 5 -GTTTCCACGCTTGGTTCAT-3 , -actin sense 5 -ATCTGGCACCACACCTTC-3 , and antisense 5 -AGCCAGGTCCAGACGCA-3 , for calibration of sample loading. Three phosphorothioate oligonucleotides of OPN were synthesized by Sangon (Shanghai, China). We used the sequences for antisense oligonucleotides, 5 -ACCACTGCCAGTCTCAT-3 ; sense oligonucleotides, 5 -ATGAGACTGGCAGTGGTT-3 ; and mismatch oligonucleotides, 5 -AACTACTATCAGTCTCGT-3 . Other chemicals and reagents were of analytical grade.
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2.3. Cell culture Adventitial fibroblasts from aortas were prepared as described [15] with modification. Briefly, rats were anesthetized with isoflurane and rapidly decapitated. The full length of the thoraco-abdominal aorta was isolated under a pool of 4 ◦ C phosphate-buffered saline (PBS) after gently opening the parietal pleura, separating 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 the use of two forceps. The remaining tissues, predominantly adventitia, were cut into small pieces and placed in 25-cm2 tissue culture flasks in DMEM containing 20% fetal bovine serum (FBS). The explants were cultivated until large colonies of cells formed surround the explants. Fibroblasts grew from these explants within 4–7 days. After achieving confluence, cells were harvested with trypsin and used for experiments at passage 3–5. The morphology and growth characteristics of the cells were typical of fibroblasts and were distinguished from smooth muscle cells by the absence of a “hill and valley” growth pattern and the lack of ␣-SM actin staining. To examine OPN mRNA expression of adventitial fibroblasts induced by UII, the cultured cells were incubated in DMEM with 10% fetal bovine serum (FBS), and then incubated with serum-free medium for 24 h. After synchronization of adventitial fibroblasts, the medium was changed to DMEM without serum. Thereafter, cells were incubated in the presence or absence of UII. To examine the molecular basis of the action of UII, we used the inhibitors for UT antagonist SB710411 (10−6 mol/l), Ca2+ channel blocker nicardipine (10−5 mol/l), PKC inhibitor H7 (10−5 mol/l), Rho protein kinase inhibitor Y-27632 (10−5 mol/l), calcineurin inhibitor CSA (10−5 mol/l) and MAPK inhibitor PD98059 (10−5 mol/l). Cells were divided into the following groups for treatment: (1) control: culture in serum-free DMEM and no UII; (2) UII: 10−10 , 10−9 , 10−8 or 10−7 mol/l UII added to the medium; (3) UII + inhibitors: pretreatment in DMEM for 0.5 h with SB710411 (10−6 mol/l), PD98059 (10−5 mol/l), CSA (10−5 mol/l), nicardipine (10−5 mol/l), H7 (10−5 mol/l), or Y-27632 (10−5 mol/l) then UII (10−8 mol/l). The cells and medium were collected after 1–24 h of incubation. 2.4. RNA isolation Total RNA was isolated directly from cells by use of Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, Trizol reagent was added directly to each dish with vigorous pipetting and then transferred to a 1.5-ml eppendorf tube. After incubation at 30 ◦ C for 5 min, 200 l of chloroform was added. The mixture was vortexed and centrifuged at 12,000 rpm for 15 min. RNA was precipitated with an equal volume of isopropanol and washed with 75% ethanol. Then, RNA was air-dried and resuspended in diethyl pyrocarbonate-treated water. The RNA preparation was treated with DNase I to remove residual traces of DNA, purified with the phenol–chloroform method and precipitated with ethanol. RNA concentration and purity were determined on a UV spectrophotometer (Bio-Rad, Hercules, CA, USA) with 260 nm absorbance and 260/280 nm absorbance ratio, respectively. Agarose gel electrophoresis (2%) and Goldview nucleic acid staining were used to examine RNA integrity. 2.5. RT-PCR Reverse transcription involved use of a commercially available kit according to the manufacturer’s instructions. The first strand-cDNA was synthesized by the Reverse Transcription System
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(Promega, Madison, USA). Briefly, 1 g of total RNA was placed in a microcentrifuge tube and incubated at 70 ◦ C for 10 min, then centrifuged briefly in a microcentrifuge, and placed on ice. The reaction was prepared by adding the following reagents (final concentration of reaction components): 5 mmol/l MgCl2 , 1× reverse transcription buffer, 1 mmol/l each dNTP mixture, 1 U/l recombinant RNasin® ribonuclease inhibitor, 15 U/g AMV reverse transcriptase, 0.5 g Oligo(dT)15 primer and nuclease-free water in a total volume of 20 l, then co-incubating according to the manufacturer’s protocol. PCR involved use of a PCR instrument (Bio-Rad, Hercules, CA, USA) with GoTaq® Green Master Mix (Promega, Madison, USA). Briefly, 1 l cDNA mixture underwent amplification in a 25 l mixture containing 12.5 l GoTaq® Green Master Mix, 11 l nuclease-free water and 0.1 mol/l each primer set. PCR conditions were initial denaturation at 95 ◦ C for 2 min, then 30 cycles of denaturation at 95 ◦ C for 0.5 min, annealing for 0.5 min at 55 ◦ C, extension at 72 ◦ C for 1 min, and a final extension at 72 ◦ C for 5 min. As an internal control for each PCR reaction, -actin cDNA was amplified for each sample under the same conditions. The PCR products were separated on 2% agarose gels and quantified with use of Quantity One 4.4.0 software (Bio-Rad, Hercules, CA, USA). Results were expressed as the ratio of OPN PCR product (174 bp) to -actin PCR product (291 bp) for each sample. All experiments were repeated 6 times.
2.6. Enzyme-linked immunosorbent assay (ELISA) OPN secretion from adventitial fibroblasts was measured by ELISA according to the manufacturer’s instructions. After incubation, the culture medium was collected, centrifuged immediately, and added to 96-well plastic plates. Absorbance of colored products was determined by use of a microplate reader set to 450 nm. Experiments were performed in duplicate and repeated 4 times.
Data analysis involved use of Prisms software. A two-tailed P value <0.05 was considered statistically significant. 3. Results 3.1. The role of OPN antisense oligonucleotide in the migration of adventitial fibroblasts induced by UII UII significantly enhanced the migration of adventitial fibroblasts (P < 0.01) as compared with control treatment (Fig. 1). The effect of UII was significantly inhibited by OPN antisense oligonucleotide (P < 0.01) but not sense or mismatch oligonucleotides as compared with UII treatment alone (P > 0.05). 3.2. The effect of UII on OPN mRNA expression in adventitial fibroblasts To validate that OPN was involved in UII actions, we examined the direct effect of UII on OPN mRNA expression. UII significantly stimulated OPN mRNA expression in adventitial fibroblasts in a time-dependent manner (Fig. 2). The effect of UII (10−8 mol/l) on OPN mRNA expression began to increase at 1 h and peaked at 3 h (all P < 0.01), then gradually decreased to the control level and lower from 12 to 24 h (P > 0.05). UII affected OPN mRNA expression in a concentration-dependent manner (Fig. 3). Maximal stimulation was reached at 10−8 mol/l UII (P < 0.01). Furthermore, the effect of UII on OPN mRNA expression was inhibited by the UT antagonist SB710411 (10−6 mol/l, P < 0.01), Rho protein kinase inhibitor Y-27632 (10−5 mol/l, P < 0.01), PKC inhibitor H7 (10−5 mol/l, P < 0.01), calcineurin inhibitor CSA (10−5 mol/l, P < 0.01), Ca2+ channel blocker nicardipine (10−5 mol/l, P < 0.01), and MAPK kinase inhibitor PD98059 (10−5 mol/l, P < 0.01) (Figs. 4 and 5), which suggests that these pathways might be involved in OPN mRNA expression induced by UII.
2.7. Cell migration assay We used antisense technology to investigate the role of OPN in migration of adventitial fibroblasts induced by UII by the transwell method (Corning), as described previously [29], which allows cells to migrate through a 8-m pore size polycarbonate membrane. Briefly, cultured adventitial fibroblasts were trypsinized, washed, and resuspended in serum-free DMEM (5 × 104 cells/ml). This suspension (500 l) was added to the lower transwell chamber so that they would be 90–95% confluent at the time of transfection. For each transfection sample, 1.0 g antisense (sense/mismatch) oligodeoxynucleotides (oligonucleotides) were diluted in 50 l DMEM, mixed with 50 l DMEM containing 3 l lipofectamine, and incubated at room temperature for 20 min. The mixture was then added to 500 l serum-free DMEM, and cells were incubated in this medium at 37 ◦ C. After 8 h, UII (10−8 mol/l) was then added to the medium for co-incubation for 24 h. Then 200 l cell suspension (1 × 105 cells/ml) was added to the upper transwell chamber. After incubation for 6 h at 37 ◦ C in the presence of 5% CO2 , the filters were removed, and cells remaining on the upper surface of the membrane were removed with a cotton swab. Then membranes were washed with PBS, and cells present beneath the membrane were fixed with 100% methanol for 2 min and stained with hematoxylin and eosin. The migration of cells was quantified by cell counts of 5 random fields at 200× magnification in each membrane. Each experiment was performed in triplicate.
2.8. Statistical analysis Results are shown as mean ± SE. Comparisons involved one-way ANOVA, followed by the Newman–Keuls multiple comparison test.
3.3. The effect of UII on OPN protein secretion from adventitial fibroblasts The effect of UII on OPN protein secretion from adventitial fibroblasts was evaluated by ELISA. UII stimulated OPN secretion in a time-dependent manner (Fig. 6). OPN secretion began to increase at 6 h and peaked at 12 h (all P < 0.05). The peak OPN protein secretion was at 10−8 mol/l UII (P < 0.01) (Fig. 7). This effect of UII on OPN protein secretion was inhibited by pretreatment with SB710411 (10−6 mol/l, P < 0.05), Y-27632, H7, CSA, nicardipine, and PD98059 (all 10−5 mol/l, all P < 0.05) (Figs. 8 and 9). 4. Discussion Recent studies suggest that both OPN and UII play critical roles in vascular remodeling [13]. In this study, we examined the role of OPN in UII-induced migration of adventitial fibroblasts and the effects of UII on OPN expression in the fibroblasts. Rat UII could induce migration of adventitial fibroblasts. This effect could be inhibited by OPN antisense but not sense or mismatch oligonucleotides, which indicates that OPN plays an important role in the migration induced by UII. Moreover, UII significantly increased both OPN mRNA and protein production in adventitial fibroblasts in a time- and concentration-dependent manner. The gene and protein expression were coincidental. Furthermore, SB710411, PD98059, nicardipine, H7, CSA and Y-27632, inhibitors or blockers of UT, MAPK, Ca2+ channel, PKC, calcineurin and Rho kinase, respectively, could inhibit the increase in OPN mRNA and protein expression induced by UII, so activation of these pathways might be involved in the UII-induced OPN biosynthesis.
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Fig. 1. Effects of antisense, sense, and mismatch oligonucleotides of OPN on adventitial fibroblast migration induced by UII. Cell migration was measured in transwell chambers. (A) Control; (B) UII; (C) UII + antisense oligonucleotides; (D) UII + sense oligonucleotides; (E) UII + mismatch oligonucleotides; (F) migrated cell numbers/field (n = 4 experiments). *P < 0.05, **P < 0.01 vs. control, ## P < 0.01 vs. UII.
Strong evidence indicates that UII is implicated in cardiovascular diseases. Specifically, the crosstalk between UII and other vascular regulators plays an important role in the pathophysiological process of the diseases, such as atherosclerosis [2,23], vascular remodeling and myocardial fibrosis [5,33,40]. Ang II and ET-1 were found to modulate UII-induced increase in myocardial distensibility [7], and UII could induce ET-1 expression in rat aortic SMCs via epidermal growth factor receptor transactivation [32]. As well, secretion of TGF-1 was upregulated in neonatal cardiac fibroblasts by UII, and TGF-1 was involved in UII-induced collagen synthesis in the cells [5]. UII also promoted TGF-1 production in rat
proximal tubular epithelial cells [30]. In the present study, we found that UII could induce the expression of OPN, an important factor in cell migration and vascular remodeling, and OPN antisense oligonucleotides could inhibit the effects of UII on migration of adventitial fibroblasts, so OPN is involved in the UII-induced migration of adventitial fibroblasts. As well, crosstalk exists
Fig. 2. UII time-dependently increased OPN mRNA expression in rat adventitial fibroblasts. Rat adventitial fibroblasts were incubated with UII (10−8 mol/l) for the times indicated. OPN mRNA expression was examined by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of ratio of OPN to -actin relative to the control group from 6 independent experiments. **P < 0.01 vs. control.
Fig. 3. UII dose-dependently increased OPN mRNA in rat adventitial fibroblasts. Rat adventitial fibroblasts were treated with UII (10−10 –10−7 mol/l) for 6 h. OPN mRNA expression was examined by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of ratio of OPN to -actin relative to the control group from 6 independent experiments (n = 6). *P < 0.05; **P < 0.01 vs. control.
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Fig. 4. UII promoted OPN mRNA expression in a UT-dependent way in rat adventitial fibroblasts. Cells were preincubated with SB710411 (SB), the UT antagonist, for 30 min, then with UII (10−8 mol/l) and the antagonist for 6 h. OPN mRNA expression was estimated by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of OPN to -actin relative to the control from 6 independent experiments. **P < 0.01 vs. control; ## P < 0.01 vs. UII.
Fig. 6. UII time-dependently increased OPN protein secretion from rat adventitial fibroblasts. Rat adventitial fibroblasts were treated with UII (10−8 mol/l) for the times indicated, the culture medium was collected, and protein level of OPN was determined by ELISA; optical density was read at 450 nm wavelength (n = 4 experiments). *P < 0.05 vs. control.
Fig. 7. UII dose-dependently increases OPN protein secretion from rat adventitial fibroblasts. Rat adventitial fibroblasts were treated with UII (10−10 –10−7 mol/l) for 12 h, the culture medium was collected, and OPN was determined by ELISA, with optical density read at 450 nm wavelength (n = 4). *P < 0.05, **P < 0.01 vs. control.
Fig. 5. Effect of different inhibitors on UII-induced OPN mRNA expression in rat adventitial fibroblasts. Cells were preincubated with Y-27632 (Y), H7, cyclosporine A (CSA), nicardipine (Ni), or PD98059 (PD; all 10−5 mol/l) for 30 min, then incubated with UII (10−8 mol/l) and the inhibitors for 6 h. OPN mRNA expression was examined by RT-PCR. Top panel: electrophoresis of RT-PCR products. Bottom panel: ratio of OPN to -actin products. Results are shown as fold increase of OPN to -actin relative to the control group from 6 independent experiments. **P < 0.01 vs. control; ## P < 0.01 vs. UII.
between UII and OPN in vascular regulation and vascular remodeling. The mechanisms of the UII effect on OPN expression are similar to the effects of UII on the induction of ET-1, TGF-1 and cell adhesion molecules. UII is able to stimulate TGF-1 secretion from neonatal cardiac fibroblasts and proximal tubular epithelial cells in rats via UT activation [5,30]. In cultured human coronary artery endothelial cells, UII increased the expression of tissue factor and cell adhesion molecules (VCAM-1/ICAM-1) through Rho kinase activation [3]. Moreover, the mechanisms involved in the UII-induced OPN expression are analogous to those of UII effects on cell migration. UII enhanced migration of human aortic SMCs through activation of an ERK-dependent pathway [22]. We previously suggested that UII may stimulate the migration of adventitial
Fig. 8. UII promotes OPN protein secretion in a UT-dependent way in rat adventitial fibroblasts. Rat adventitial fibroblasts were preincubated with UT antagonist SB710411 (SB; 10−6 mol/l) for 30 min, then incubated with UII (10−8 mol/l) and the antagonist for 12 h. The culture medium was then collected, and OPN was determined by ELISA, with optical density read at 450 nm wavelength (n = 4). *P < 0.05 vs. control, # P < 0.05 vs. UII.
fibroblasts through PKC, MAPK, calcineurin, and Rho kinase signal transduction pathways [41]. Recently, UII was found to induce migration of endothelial progenitor cells via activation of the RhoA/Rho kinase pathway [36]; UII could induce OPN expression via the UT, Ca2+ channel, PKC, MAPK, calcineurin and Rho kinase pathways, which provided new insights into UII actions. In addition, UII was found to induce adventitial fibroblast proliferation, collagen synthesis and secretion, and phenotypic differentiation through the activation of UT, PKC, MAPK, calcineurin, Rho protein kinase
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Fig. 9. Effects of different inhibitors on UII-induced OPN protein secretion in rat adventitial fibroblasts. Rat adventitial fibroblasts were preincubated with H7, cyclosporine A (CSA), Y-27632 (Y), PD98059 (PD), or nicardipine (Ni; all 10−5 mol/l) for 30 min, then incubated with UII (10−8 mol/l) and the inhibitors for 12 h. The culture medium was collected, and OPN was determined by ELISA; optical density was read at 450 nm wavelength (n = 4). **P < 0.01 vs. control; # P < 0.05, ## P < 0.01 vs. UII.
and/or calcium channel [39,41]. These signal pathways may play important roles in the activation of adventitial fibroblasts induced by UII. Vascular adventitial fibroblasts play a key role in vascular remolding through phenotypic conversion, proliferation and migration. Numerous cytokines and factors may influence these processes [19,25]. Both OPN and UII are involved in atherosclerotic plaque and neointima formation after angioplasty [2,13,37]. OPN also enhanced the migratory ability of cultured aortic adventitial fibroblasts from spontaneously hypertensive rats [29]. Fibroblast growth factor 1 could upregulate OPN expression via Src/MEK/MAP kinase signaling pathways in rat aortic SMCs, with further mediation of the migration of adventitial fibroblasts [16]. Recently, the autocrine expression of OPN was found to play a major role in SMC migration induced by platelet-derived growth factor [14]. Our previous study demonstrated that UII could induce phenotypic conversion and migration of vascular adventitial fibroblasts as an autocrine and paracrine factor [39,41]. The present study indicates that UII effects on vascular remodeling may partly involve the autocrine expression of OPN in the adventitial fibroblasts. In conclusion, we revealed that UII-induced migration of adventitial fibroblasts could be inhibited by OPN antisense oligonucleotides. In addition, UII significantly increased both the mRNA and protein expression of OPN in adventitial fibroblasts, via the activation of the UT, and the Ca2+ channel, MAPK, PKC, calcineurin and Rho kinase signal transduction pathways. This study provides new insights into UII actions that UII may act partly via the autocrine production of OPN in the adventitial fibroblasts. Simultaneous blockade of UII and of OPN expression could be a promising therapeutic strategy to limit vascular remodeling. Acknowledgments This project was supported by National Natural Science Foundation of China (No. 30971273) and the Natural Science Foundation of Guangdong Province (No. 9151051501000016) of China. The experiment was completed with the assistance from the Laboratory of Molecular Biology & Cardiology, First Affiliated Hospital, Shantou University Medical College. References [1] Abdel-Azeez HA, Al-Zaky M. Plasma osteopontin as a predictor of coronary artery disease: association with echocardiographic characteristics of atherosclerosis. J Clin Lab Anal 2010;24:201–6.
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