Research Paper

Vitamin D Analogs Modulate the Expression of Plasminogen Activator Inhibitor-1, Thrombospondin-1 and Thrombomodulin in Human Aortic Smooth Muscle Cells
J. Ruth Wu-Wong, Masaki Nakane, Junli Ma

Abbott Laboratories, Abbott Park, Ill., USA

Address of Corresponding Author

J Vasc Res 2007;44:11-18 (DOI: 10.1159/000097812)

 Outline

• Key Words
• Abstract
• Introduction
• Materials and Methods
• Materials
• Cell Cultures
• Real-Time Reverse Transcription Polymerase Chain Reaction
• SDS-PAGE and Western Blot Analysis
• Results
• Discussion
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Vitamin D receptor
• Cardiovascular disease
• Plasminogen activator inhibitor-1
• Thrombospondin-1
• Thrombomodulin

  Abstract

Background/Aims: Plasminogen activator inhibitor-1 (PAI-1), thrombospondin-1 (THBS1) and thrombomodulin (TM) are involved in atherothrombosis. Vitamin D receptor agonists (VDRAs) provide survival/cardiovascular benefits for chronic kidney disease patients. Methods: The effects of VDRAs on regulating PAI-1, THBS1 and TM in human aortic smooth muscle cells (SMC) and endothelial cells (EC) were studied. Results: In SMC, paricalcitol and calcitriol downregulated the expression of PAI-1 mRNA and protein in a dose-dependent manner (EC₅₀ = 0.7 and 4.4 nM, respectively). Both drugs also downregulated THBS1 mRNA and protein (EC₅₀ = 1.6 and 3.9 nM, respectively). In contrast, paricalcitol and calcitriol upregulated TM mRNA and protein (EC₅₀ = 28.9 and 25.5 nM, respectively). EC did not express VDR, and VDRAs failed to induce CYP24A1, a VDR target gene. The effect of paricalcitol on THBS1 in SMC was blocked by cycloheximide, while its effect on TM and CYP24A1 was not affected, suggesting that the regulation of THBS1 by VDR may be mediated through intermediate factors, but that TM is likely a direct target of VDR. Conclusion: VDR may play a role in atherothrombosis via regulation of PAI-1, THBS1 and TM.

Copyright © 2007 S. Karger AG, Basel

 Introduction

The steroid hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol] activates multiple signaling pathways in various cells and tissues. Although the synthesis of vitamin D₃ occurs naturally in the skin with adequate sunlight exposure, vitamin D₃ is not active and needs to be converted to 25(OH)D₃ by 25-hydroxylase in the liver. From the liver, 25(OH)D₃ is transported to the kidney and hydroxylated by 25-hydroxyvitamin D-1-hydroxylase to form the active hormone, calcitriol [1]. Calcitriol is metabolized by 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) [2] to yield the biliary excretory product calcitroic acid. The binding of 1,25(OH)₂D₃ or its analogs to the vitamin D receptor (VDR), a nuclear receptor, activates VDR to recruit cofactors to form the VDR/cofactor complex, which binds to vitamin D response elements in the promoter region of target genes to regulate gene transcription [3].

Chronic kidney disease (CKD) patients experience a high mortality rate from cardiovascular disease [4, 5]. Vitamin D analogs (or VDR agonists, VDRAs) such as paricalcitol and calcitriol that activate VDR are commonly used to manage secondary hyperparathyroidism associated with CKD [6]. Recent retrospective clinical observations show that VDRAs provide survival benefit for stage 5 CKD patients in the effectiveness order of paricalcitol > calcitriol > no VDRA therapy, independent of the parathyroid hormone and calcium levels [7,8,9]; the survival benefit is associated with a decrease in cardiovascular-related incidents [10]. More recent studies using the time-dependent analyses suggest that the survival benefit is in the order of intravenous paricalcitol > intravenous calcitriol > oral vitamin D analogs = no vitamin D analogs [11, 12].

Although data from clinical studies demonstrate the positive impact of VDRAs on the cardiovascular system, the mechanism of action is largely unknown. CKD patients are known to have advanced atherosclerotic disease [13,14,15]. Atherosclerosis, the principal cause of myocardial infarction, stroke and peripheral vascular disease, is a process that involves a complex interplay among different factors such as inflammation, thrombosis and various cell types including smooth muscle cells (SMC) and endothelial cells (EC). Atherosclerosis is asymptomatic during a long period and dramatically changes its course when complicated by thrombosis. Atherothrombosis and the rupture of vulnerable plaques are responsible for coronary thrombosis, the main cause of unstable angina, acute myocardial infarction, and sudden cardiac death [16]. Under normal conditions, many factors involved in atherothrombosis are predominantly localized in EC, but vascular injury often results in increased expression of these factors in SMC [17,18,19,20,21].

We have previously shown that in primary culture of human coronary artery SMC, VDR is involved in modulating the expression of plasminogen activator inhibitor-1 (PAI-1) [22], one of the risk markers for coronary heart disease [23]. It is known that thrombomodulin (TM), a monomeric transmembrane protein that serves as a cell surface receptor for thrombin [24], and thrombospondin-1 (THBS1), a large glycoprotein that is released into the extracellular matrix by several cell types including SMC [25], also play roles in fibrinolysis and thrombogenicity. In this study, we investigated the effects of VDRAs on modulating PAI-1, THBS1 and TM expression in primary culture of human aortic SMC and EC.

 Materials and Methods

 Materials

1,25-dihydroxyvitamin D₃ (calcitriol) and 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol) were from Abbott Laboratories. Drugs were dissolved in ethanol to make a 10-mM stock solution. Other reagents were of analytical grade.

 Cell Cultures

Primary culture of human aortic SMC (Cambrex, Walkersville, Md., USA) was grown in SmGM-2 containing 5.5 mM glucose, 5% FBS, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, 5 µg/ml insulin, 2 ng/ml hFGF, and 0.5 ng/ml hEGF at 37°C in humidified 5% CO₂/95% air. Cells were grown to >80% confluence and used within five passages. Primary culture of human aortic EC (Cambrex) was grown in EGM-2 Bullet Kit medium containing 2% FBS (Cambrex) at 37°C and used within four passages.

 Real-Time Reverse Transcription Polymerase Chain Reaction

Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed with an iCycler (BioRad, Hercules, Calif., USA). Each sample has a final volume of 25 µl containing 100 ng of cDNA, 0.4 µM each of the forward and reverse PCR primers and 0.1 µM of the TaqMan^TM probe for the gene of interest (Applied Biosystems). Temperature conditions consisted of a step of 5 min at 95°C, followed by 40 cycles of 60°C for 1 min and 95°C for 15 s. Data were collected during each extension phase of the PCR reaction and analyzed with the software package (BioRad). Threshold cycles were determined for each gene.

 SDS-PAGE and Western Blot Analysis

Cells (1 × 10⁶ cells per sample) pretreated with or without test agents were solubilized in 50 µl of SDS-PAGE sample buffer (Invitrogen, Carlsbad, Calif., USA), and the protein content in each sample was determined by the Pierce (Rockford, Ill., USA) bicinchoninic acid protein assay. Samples were resolved by SDS-PAGE using a 4-12% NuPAGE gel (Invitrogen), and proteins were electrophoretically transferred to polyvinylidene fluoride membrane for Western blotting. The membrane was blocked for 1 h at 25°C with 5% nonfat dry milk in PBS Tween-20 (PBS-T) and then incubated with a mouse anti-PAI-1 monoclonal antibody (1,000-fold dilution; Santa Cruz Biotechnology, Santa Cruz, Calif., USA), a mouse anti-THBS1 monoclonal antibody (2,000-fold dilution; Calbiochem, La Jolla, Calif., USA), or a mouse anti-TM monoclonal antibody (2,000-fold dilution; Santa Cruz Biotechnology) in PBS-T overnight at 4°C. The membrane was washed with PBS-T and incubated with a horseradish peroxidase-labeled anti-mouse antibody for 1 h at 25°C. The membrane was then incubated with detection reagent (SuperSignal WestPico, Pierce). Specific bands were visualized by exposing the membrane to Kodak BioMax films. Band intensity was quantified by Quantity One (BioRad). For VDR and retinoid X receptor (RXR) detection, membranes were incubated with a rabbit anti-VDR (Active Motif, Carlsbad, Calif., USA) or RXR (Santa Cruz Biotechnology) polyclonal antibody (500-fold dilution), followed by a horseradish peroxidase-labeled anti-rabbit antibody.

 Results

Results from real-time RT-PCR analysis show that the PAI-1 mRNA level was reduced by both paricalcitol and calcitriol in a dose-dependent manner in SMC (fig. 1a). The maximal suppression of PAI-1 mRNA expression (approximately 60%) was achieved at 0.1 µM of paricalcitol or calcitriol. The EC₅₀ values (concentration that achieves 50% of maximum effect) of paricalcitol and calcitriol on inhibiting PAI-1 mRNA were 0.7 and 4.4 nM, respectively. Figure 1b shows that paricalcitol and calcitriol, analyzed by Western blotting, downregulated the expression of PAI-1 protein in a dose-dependent manner. These results are consistent with our previous observations in human coronary artery SMC that vitamin D analogs downregulated PAI-1 expression [22].

Fig. 1. Effect of paricalcitol and calcitriol on PAI-1 in SMC. a SMC in serum-free medium were treated with paricalcitol or calcitriol at indicated concentrations for 24 h. RNA was isolated using Trizol (Invitrogen) followed by a Qiagen RNeasy mini kit. Real-time RT-PCR was performed as described in Materials and Methods. The expression level of PAI-1 mRNA was first normalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA level and then calculated as percent of control (untreated, 100%). Values shown are means ± standard deviations (n = 4). b Cells were treated with paricalcitol and calcitriol at indicated concentrations for 48 h. Cells were then solubilized and the protein content in each sample was determined by the Pierce bicinchoninic acid protein assay. Western blotting was performed as described in Materials and Methods. The specific bands were visualized and the density of each band was measured. The band was first normalized to the protein content in each sample and then calculated as percent of control (untreated, 100%). Values shown in the bar graph are means ± standard deviations (n = 3). The band picture shown is representative of 3 independent experiments. C = Control, no drug treatment. Statistical comparisons were performed by ANOVA. * p < 0.05, ** p < 0.01.

Similarly, the THBS1 mRNA level was reduced by both paricalcitol and calcitriol in a dose-dependent manner (fig. 2a). The maximal suppression of THBS1 mRNA expression (approximately 50%) was achieved at 0.1 µM of paricalcitol or calcitriol. The EC₅₀ values of paricalcitol and calcitriol on suppressing THBS1 mRNA were 1.6 and 3.9 nM, respectively. Figure 2b shows that paricalcitol and calcitriol downregulated the expression of THBS1 protein in a dose-dependent manner.

Fig. 2. Effect of paricalcitol and calcitriol on THBS1 in SMC. a SMC in serum-free medium were treated with paricalcitol or calcitriol at indicated concentrations for 24 h. Samples were processed for real-time RT-PCR and data analysis done as in figure 1a. Values shown are means ± standard deviations (n = 4). b Cells were treated with paricalcitol and calcitriol at indicated concentrations for 48 h. Samples were processed for Western blotting and data analysis was done as in figure 1b. Values shown are means ± standard deviations (n = 3). C = Control, no drug treatment. Statistical comparisons were performed by ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001.

In comparison, results from real-time RT-PCR analysis showed that the TM mRNA level was upregulated by both paricalcitol and calcitriol in a dose-dependent manner in SMC (fig. 3a). At 0.1 µM, paricalcitol and calcitriol stimulated an increase in the TM mRNA level by 4.9 and 6.0 fold, respectively. The EC₅₀ values of paricalcitol and calcitriol on upregulating TM mRNA were 28.9 and 25.5 nM, respectively. Consistent with the real-time RT-PCR results, figure 3b shows that paricalcitol and calcitriol increased the expression of TM protein in a dose-dependent manner.

Fig. 3. Effect of paricalcitol and calcitriol on TM in SMC. a SMC in serum-free medium were treated with paricalcitol or calcitriol at indicated concentrations for 24 h. Samples were processed for real-time RT-PCR and data analysis was done as in figure 1a. Values shown are means ± standard deviations (n = 4). b Cells were treated with paricalcitol and calcitriol at indicated concentrations for 48 h. Samples were processed for Western blotting and data analysis was done as in figure 1b. Values shown are means ± standard deviations (n = 3). C = Control, no drug treatment. Statistical comparisons were performed by ANOVA. * p < 0.01, ** p < 0.001.

Interestingly, studies comparing SMC and EC indicated paricalcitol at 1-100 nM did not exhibit a significant effect on the expression of PAI-1 or TM in EC (data not shown). The results were consistent with our previous report that VDRAs, in human coronary artery EC, do not affect the expression of PAI-1 due to the absence of VDR [22].

Indeed, when we examined the expression of VDR, no VDR was detected in human aortic EC (fig. 4a). As a control, RXR was detected in these cells. Figure 4a also shows that SMC expressed VDR (molecual weight 55 kDa), which was increased by paricalcitol in a dose-dependent manner (a 3.5-fold increase observed at 0.1 µM paricalcitol). RXR was also detected in SMC, but its expression level was not affected by paricalcitol treatment. As expected, figure 4b shows that paricalcitol profoundly stimulated the expression of CYP24A1 mRNA in a dose-dependent manner in SMC, but failed to have an inducing effect on CYP24A1 mRNA in EC. These results confirm that primary culture of human aortic EC, similar to coronary artery EC, do not express a functional VDR, and paricalcitol fails to evoke a functional response.

Fig. 4. VDR/RXR expression and effect of paricalcitol on CYP24A1 mRNA in SMC and EC. a Cells in serum-free medium were treated with paricalcitol at indicated concentrations for 48 h. Western blotting was performed as in figure 1. Results shown are representative of two independent experiments. C = Control, no drug treatment. b Cells were treated with paricalcitol at indicated concentrations for 24 h, and RNA was isolated for real-time RT-PCR. The expression level of CYP24A1 mRNA was normalized to the glyceraldehyde-3-phosphate dehydrogenase mRNA level. Values shown are means ± standard deviations (n = 4). Statistical comparisons were performed by ANOVA. * p < 0.01.

Previously, we have shown that the effect of paricalcitol on suppressing PAI-1 in human coronary artery SMC was blocked by cycloheximide, suggesting that protein synthesis was involved. Here, we investigated the mechanism for VDR-mediated THBS1 suppression and TM upregulation in SMC. Aortic SMC were treated with 3 µg/ml cycloheximide for 4 h before addition of paricalcitol. Table 1 shows that in cells treated with cycloheximide, the suppression of THBS1 by paricalcitol was no longer statistically significant, while paricalcitol induced TM mRNA in a dose-dependent manner and the effects were not significantly affected by cycloheximide (EC₅₀ = 10.6 and 11.7 nM in the presence or absence of cycloheximide, respectively). As a control, paricalcitol induced CYP24A1 mRNA in a dose-dependent manner, and the induction was not significantly affected by cycloheximide (EC₅₀ = 2.3 and 4.9 nM in the presence or absence of cycloheximide, respectively).

Table 1. Effect of paricalcitol on THBS1 and TM expression differentially blocked by cycloheximide

 Discussion

PAI-1, THBS1 and TM are known to be involved in fibrinolysis and thrombogenicity. Upregulation of PAI-1 and THBS1 is associated with the development of thrombosis, atherosclerosis and vascular injury [26, 27], while an early loss of TM expression resulted in an enhanced thrombin generation [24, 28], and a local overexpression of TM prevented atherothrombosis [29]. Previously, it has been shown that TM mRNA expression is decreased in aorta, liver and kidney prepared from the VDR knockout mice [30], and calcitriol upregulates TM expression in human leukemic cells and monocytes [31, 32]. Recently, THBS1 has also been shown as an anti-angiogenic agent [27], but the role of VDR in the regulation of THBS1 in human SMC has not been reported before.

We have previously reported that human coronary artery EC do not express a functional VDR at 55 kDa, and both paricalcitol and calcitriol downregulated the expression of PAI-1 mRNA and protein in human coronary artery SMC [22]. A review of the literature shows that there are inconsistent results regarding whether EC express VDR, and also about the effect of vitamin D analogs on PAI-1 expression. For example, it has been reported that paricalcitol suppresses the expression of PAI-1 protein, while calcitriol stimulates the expression of PAI-1 in human umbilical vein EC [33]. It is possible that cells of different origins may differ in VDR expression and in their responses to vitamin D analogs. Therefore, it is important to study SMC and EC prepared from different types of tissues in order to develop a better understanding of the role of VDR in the vasculature. Our current study demonstrates that human aortic EC do not express VDR either. Furthermore, we found that VDR plays a role in downregulating the expression of PAI-1 not only in coronary artery SMC, but also in aortic SMC. In addition, we have extended the studies to include TM and THBS1. Our results unequivocally show that VDRAs upregulate TM and downregulate THBS1 in human aortic SMC.

Our study finds that aortic EC do not express VDR, and VDRAs fail to induce CYP24A1 mRNA expression in these cells, which is consistent with our previous report that human coronary artery EC do not express VDR [22]. Although under normal conditions many of these factors involved in atherothrombosis are predominantly localized in EC, vascular injury often results in altered expression patterns of these factors. For example, in both human and mouse, the expression of PAI-1 is prevalent in activated EC at the early stages of atherosclerosis, but its expression in advanced atherosclerotic lesions is predominant in SMC [17]. In atherosclerotic vessels, TM has been shown to be markedly downregulated in EC, and SMC may become a relevant source of TM under pathological conditions such as advanced atherosclerosis [18,19,20]. THBS1 expression in the vascular wall is significantly increased in injured vessels and in stent-induced neointima [34]. Furthermore, increased expression of THBS1 has been detected in the adventitia of blood vessels from diabetic rats and in cultured vascular SMC in response to glucose stimulation [21].

The effect of paricalcitol on suppressing THBS1 in aortic SMC is blocked by cycloheximide, while the upregulation of TM by paricalcitol is not. In the control study, the effect of paricalcitol on inducing the CYP24A1 gene, which is known to be a direct target gene of VDR, is not affected by cycloheximide treatment either. This result suggests that the regulation of THBS1 by VDR may be mediated through intermediate factors involving protein synthesis, but TM is likely a direct target gene of VDR. Our previous studies in coronary artery SMC show that VDR-mediated suppression of PAI-1 can also be blocked by cycloheximide. These results suggest that at least in the case of CYP24A1 and TM, VDR may directly bind to VDR response elements in the promoter region of target genes to regulate gene expression. However, when VDR is involved as a negative regulator such as in the case of PAI-1 and THBS1, an indirect mechanism may be involved. More studies are required to elucidate the molecular mechanism of how VDR mediates the suppression of PAI-1 and THBS1.

In healthy individuals, the average level of calcitriol is approximately 45 pg/ml (equivalent to about 0.1 nM), while the precursor, 25(OH)D₃, is at 29.6 ng/ml (about 74 nM) [35]. Calcitriol suppresses the expression of parathyroid hormone with 50% inhibitory concentration in the sub-nM range [36], which is consistent with its endocrine function in mineral homeostatsis and bone metabolism. However, we have observed in this study that calcitriol modulates the expression of PAI-1, THBS1 and TM in SMC with EC₅₀ in the 1- to 30-nM doses which are 10- to 300-fold higher than the serum level. Although it is beyond the scope of this study, these data seem to suggest that calcitriol may act as a paracrine/autocrine hormone in the vasculature. Previously, Schwartz et al. [37] showed that human prostate cells synthesize calcitriol from 25(OH)D₃. More studies are required to investigate whether the vascular cells also express 25-hydroxyvitamin D-1-hydroxylase to produce a high concentration of calcitriol locally.

In summary, in this report, we show that VDRAs modulate the expression of PAI-1, THBS1 and TM in human aortic SMC. Our data also suggest that TM is a direct target gene of VDR, and the regulation of THBS1 by VDR is likely mediated through intermediate factors. Although more studies are required to clarify whether VDR is involved in modulating vascular function, our data suggest that the involvement of the VDR in regulating PAI-1, THBS1 and TM in SMC may account for some of the factors contributing to the benefits of VDRA therapy on reducing mortality and morbidity risk in stage 5 CKD patients.

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  Author Contacts

Dr. J. Ruth Wu-Wong
Abbott Laboratories, R46R, AP9
100 Abbott Park Rd.
Abbott Park, IL 60064 (USA)
Tel. +1 847 938 8492, Fax +1 847 936 1550, E-Mail ruth.r.wuwong@abbott.com

  Article Information

Received: July 15, 2006
Accepted after revision: September 28, 2006
Published online: December 7, 2006
Number of Print Pages : 8
Number of Figures : 4, Number of Tables : 1, Number of References : 37

  Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 44, No. 1, Year 2007 (Cover Date: January 2007)

Journal Editor: Pohl, U. (Munich)
ISSN: 1018-1172 (print), 1423-0135 (Online)

For additional information: http://www.karger.com/JVR

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.