Dexamethasone Downregulates Calcification-Inhibitor Molecules and Accelerates Osteogenic Differentiation of Vascular Pericytes
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John Paul Kirton, Fiona L. Wilkinson, An
参见附件。
the Wellcome Trust Centre for Cell-Matrix Research and Division of Cardiovascular and Endocrine Sciences, Faculty of Medical and Human Sciences, University of Manchester, UK.
Abstract
Vascular calcification is present in many pathological conditions and is recognized as a strong predictor of future cardiovascular events. Current evidence suggests that it is a regulated process involving inducing and inhibitory molecules. Glucocorticoids have great clinical importance as antiinflammatory drugs and can act as potent inducers of osteogenic differentiation in vitro. The effect of glucocorticoids on vascular cells in vivo remains obscure. Pericytes are pluripotent cells that can differentiate into osteoblasts, and recent evidence suggests that they could participate in vascular calcification. We hypothesized that the synthetic glucocorticoid dexamethasone would enhance the rate of pericyte differentiation and mineralization in vitro with a concomitant suppression of calcification-inhibitory molecules. Three weeks of dexamethasone treatment induced a 2-fold increase in (1) alkaline phosphatase activity, (2) calcium deposition, and (3) the number of nodules formed in vitro; and a reduction in the expression of matrix Gla protein (MGP), osteopontin (OPN), and vascular calcification-associated factor (VCAF) mRNAs. The glucocorticoid receptor antagonist Org 34116 abolished dexamethasone-accelerated pericyte differentiation, nodule formation, and mineralization. Data obtained using Org 34116, the transcription inhibitor actinomycin D, and the protein synthesis inhibitor cyclohexamide suggest that MGP, OPN, and VCAF mRNA abundance are controlled at different and multiple levels by dexamethasone. This is the first report showing that dexamethasone enhances the osteogenic differentiation of pericytes and downregulates genes associated with inhibition of mineralization. Our study highlights the need for further investigation into the long-term consequences of prolonged glucocorticoid therapy on vascular calcification.
Key Words: pericytes differentiation calcification gene regulation glucocorticoid
Introduction
Calcification can lead to devastating clinical consequences when present in blood vessels. It has been suggested that cells maintain a balance between calcification inducing and inhibitory proteins in soft tissues in such a way that the deposition of a mineralized matrix is normally avoided. Alterations in this balance may be brought about by injury or disease, with the subsequent deposition of calcific deposits in the vessel wall. It is now well established that vascular calcification is associated with elevated expression of bone-related proteins1,2 and that pericyte-like cells exist in the vasculature, which, under appropriate conditions, can be induced to display characteristics of the osteoblastic lineage.3,4 Our previous studies have demonstrated that cultured postconfluent pericytes form large calcified multicellular nodules both in vitro and in vivo,5 the matrix of which resembles that found in calcified atherosclerotic plaques and bone.5 Although factors such as transforming growth factor (TGF-),6 tumor necrosis factor (TNF-),7 and acetylated LDL8 have been shown to be involved in the promotion of calcification in vitro, the underlying mechanisms of vascular calcification have not been fully elucidated.
The main risk factors for atherosclerosis include not only the classic factors such as age, high cholesterol levels, and hypertension but also prolonged glucocorticoid therapy. The mechanisms underlying the adverse effects of glucocorticoid excess on the vascular system are complex and have not yet been fully explained. It is well recognized that vascular ossification and osteoporosis often coexist in patients, whereby bone mineral is formed within the vasculature, while it is simultaneously lost from the skeleton.9 How this buildup and loss of mineral can occur simultaneously in patients remains to be elucidated.
Glucocorticoids are a class of steroid hormones produced by the cortex within the mammalian adrenal gland and are normally maintained at steady-state levels. A deregulation of endogenous levels is induced by vascular injury in vivo10 and could be a contributing factor in the development of vascular calcification. The synthetic glucocorticoid dexamethasone induces an osteoblastic differentiation pathway in many different cell types in vitro, including human bone marrow–derived stromal cells,11,12 fetal rat calvarial cells,13 bovine vascular smooth muscle cells (SMCs),14 and mouse embryo–derived NIH3T3 fibroblasts.15 This study tests the hypothesis that dexamethasone enhances the in vitro osteoblastic differentiation of vascular pericytes and that this may be associated with a decrease in the expression of calcification-inhibitor molecules. We investigate several parameters within pericytes exposed to dexamethasone, in terms of their proliferation, the time-course of nodule formation and mineralization, the expression of osteoblastic markers, and the mechanisms underlying these effects.
Materials and Methods
Cell Culture and Nodule Formation
Pericytes were isolated from bovine retinas essentially as previously described,16 except that cells were isolated and subsequently maintained either in the continued presence or absence of dexamethasone (10 nmol/L) in Eagle’s minimal essential medium (MEM) containing 20% FCS, 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 50 μg/mL ascorbate-2-phosphate, and nonessential amino acids. Control cells were treated with vehicle alone. For experiments, pericytes were plated at 1.5x104 cells/cm2, with a media change every 4 days. In some experiments, the glucocorticoid receptor (GR) antagonist Org 34116 (1 μmol/L; a kind gift from Angela Fisher, Organon, Motherwell, Scotland, UK) was also included, and the cells were subsequently harvested at weekly time points. The area occupied by nodules was determined using AnalySIS software. Mineralized nodules were visualized by washing in PBS, fixing in 4% formaldehyde, and staining with 1% (wt/vol) Alizarin red S (pH 4.2). Actinomycin D and cyclohexamide were used at a final concentration of 5 μg/mL and 35 mmol/L, respectively. Cells were grown for 3 weeks to ensure detectable baseline levels of vascular calcification-associated factor (VCAF), osteopontin (OPN), and matrix Gla protein (MGP) mRNA and protein; then actinomycin D or cyclohexamide was added to the media for 8 and 4 hours, respectively, before harvesting.
Alkaline Phosphatase Activity
Cells were seeded in 6-well plates and maintained up to 4 weeks. Proteins were extracted at weekly time points by freeze-thawing the cells in 0.05% Triton X-100 in PBS. Total cellular proteins were measured using Bradford’s assay (Bio-Rad). Alkaline phosphatase (ALP) activity was quantified in protein lysates (15 μg) made up to 100 μL in 2-amino-2-methyl 1-propanol (AMP) buffer containing 8 mmol/L -nitrophenylphosphate (-NPP) (Sigma-Aldrich) as substrate and incubated at 37°C for 2 hours before terminating the reaction with 0.5 mol/L NaOH (100 μL). The absorbance was measured at 405 nm and ALP activity calculated as nmol/L -nitrophenol converted per microgram of protein per minute.
Calcium Assay
45Ca deposition was assayed as described.17 Cells were seeded in 24-well plates and harvested weekly up to 4 weeks. 45CaCl2 (ICN) was added (1.0 μCi/mL for 48 hours), and the cells were harvested in PBS (0.3 mL), placed into vials containing perchloric acid (0.2 mL) with 3% hydrogen peroxide (0.3 mL), and incubated at 80°C for 1 hour. Samples were then dissolved in ethylene glycol monoethyl ether (0.6 mL), and the radioactivity was quantified by scintillation counting.
RNA Analysis
Cells were seeded in 25-cm2 flasks and the RNA harvested weekly using TRIzol reagent (Invitrogen). Northern blot analysis and probe preparation were performed as previously described.5,18 Densitometry was used to express the mRNA abundance of MGP, OPN, and VCAF relative to 18S ribosomal RNA. To determine mRNA stability, total RNA was harvested at selected time points following incubation with the transcriptional inhibitor actinomycin D. mRNA abundance at the time of addition of actinomycin D was set at 100%, and the rate of OPN, MGP, and VCAF mRNA decay was compared in dexamethasone- versus vehicle-treated cells.
Western Blot and Immunocytochemical Analysis
Whole cell lysates were extracted as previously described,16 and cytosolic and nuclear fractions were separated using a BioVision kit (Mountain View, Calif) according to the protocol of the manufacturer. Proteins (20 μg/lane) were separated by SDS-PAGE (10% gels) and transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were incubated with a nuclear factor B (NF-B) antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif) at 4°C for 16 hours and a horseradish peroxidase–conjugated swine anti-rabbit secondary antibody (1:1000 dilution) and developed as described.16 For immunocytochemistry, pericytes were cultured in chamber slides for 48 hours, fixed in 100% methanol, and blocked in 5% goat serum/PBS for 1 hour at room temperature. The NF-B polyclonal antibody (1:200 dilution) was used with a goat anti-rabbit IgG, conjugated to AlexaFluor 488 secondary antibody (Molecular Probes; 1:300 dilution). Cells were counterstained with 4 μg/mL 4',6-diamidino-2-phenylindole (DAPI).
Data Analysis
The data for the ALP, nodule formation, and 45Ca assay are shown as the mean±SD of 2 experiments performed in triplicate wells. An unpaired, 2-tailed Student’s t test or ANOVA was used to determine statistical significance between mean values, as appropriate. A value of P<0.05 was considered statistically significant. For Northern and Western blots, data are representative of at least 2 experiments.
Results
Dexamethasone Induces Differentiation and Mineralization of Pericytes In Vitro
Initially, the effects of continued presence or absence of dexamethasone on pericyte proliferation, alkaline phosphatase activity, calcium incorporation, and mineralized nodule formation were investigated. Using a thymidine incorporation assay, pericyte growth was investigated during the first week of culture, and dexamethasone was found to have no significant effect on pericyte proliferation compared with controls (data not shown).
There was a 4.4-fold increase in ALP activity in pericytes after dexamethasone treatment during 3 weeks of culture compared with 2.1-fold increase in the vehicle-treated cells (Figure 1). At week 3, there was a 2.2-fold increase in ALP activity in cells grown in the presence of dexamethasone compared with vehicle-treated cells (Figure 1). No further increase in ALP activity was detected at week 4.
Nodule formation was detected as early as 1 week after plating in the presence of dexamethasone (Figure 2A), culminating in a 2.9-fold increase in the number of nodules formed, compared with vehicle-treated cells at week 4 (Figure 2B). In addition, nodules were larger (Table) and mineralized earlier in the presence of dexamethasone compared with controls (Figure 2A). Furthermore, in the presence of the glucocorticoid antagonist Org 34116, the dexamethasone-induced increase in pericyte nodule number and size was abolished (Table).
Culturing pericytes for 4 weeks in dexamethasone increased 45Ca deposition 2.4-fold compared with vehicle-treated cells (Figure 3). The increased calcium incorporation in the dexamethasone-treated cells was also abolished in the presence of Org 34116 (Figure 3).
Dexamethasone Reduces OPN, MGP, and VCAF mRNA Abundance in Pericytes
MGP, OPN, and VCAF mRNAs were examined in cells maintained in the presence and absence of dexamethasone. As reported previously, we detected an increase in expression of OPN,5 MGP, and VCAF mRNA18 in vehicle-treated control cells with increasing time in culture (Figure 4). All 3 genes were also upregulated in pericytes cultured in the presence of dexamethasone for 2 to 3 weeks (Figure 4); however, dexamethasone caused a decrease in the mRNA abundance of all 3 transcripts compared with vehicle-treated cells (Figure 4), with the decrease in MGP and OPN mRNA being particularly striking (Figure 4B and 4C). These differences in gene expression were maintained when pericytes were cultured with and without dexamethasone for 4 weeks (results not shown).
Control of MGP, OPN, and VCAF Gene Expression by Dexamethasone
The decline in abundance of MGP, OPN, and VCAF mRNAs could be caused directly by either a suppression of transcription or accelerated degradation of RNA or indirectly by the involvement of a second transcription factor. mRNA stability was investigated using the transcription inhibitor actinomycin D. MGP, OPN, and VCAF mRNAs were found to have long half-lives of more than 8 hours, which were not altered by culturing cells in the presence of dexamethasone (data not shown), suggesting that dexamethasone had not induced degradation of these mRNAs.
To investigate transcriptional control mediated by dexamethasone binding to its receptor, the cells were treated with Org 34116. Pericytes were divided into 3 groups: (1) without dexamethasone (–), (2) with dexamethasone (+), and (3) in the presence of dexamethasone plus Org 34116. The presence of Org 34116 blocked the dexamethasone-induced downregulation of MGP and VCAF mRNAs (Figure 5A, 5B, and 5D), adding support for a transcriptional mechanism of regulation of these genes by dexamethasone. However, the GR antagonist had no effect on OPN expression after dexamethasone treatment (Figure 5A and 5C), indicating that dexamethasone regulates OPN gene expression by a different mechanism to that shown for MGP and VCAF.
Because OPN expression was unaffected by the GR antagonist, it is likely that another protein is involved in the regulation of OPN by dexamethasone. To investigate this possibility and to assess the role of de novo protein synthesis in the downregulation of MGP, VCAF, and OPN mRNA during continuous exposure to dexamethasone, pericytes were treated with the protein synthesis inhibitor cyclohexamide for 4 hours (Figure 6). As previously shown, dexamethasone downregulated expression of MGP and OPN mRNA compared with control cells. However, the mRNA abundance of both MGP and OPN was found to be unaffected by cyclohexamide treatment (Figure 6). In contrast, cyclohexamide abolished the modest downregulation of VCAF mRNA expression after exposure of the cells to dexamethasone, suggesting involvement of another protein in VCAF regulation by dexamethasone (Figure 6).
Dexamethasone Prevents Nuclear Accumulation of NF-B in Pericytes
Growing evidence suggests that the antiinflammatory effects of glucocorticoids are mediated by a downregulation of NF-B. Therefore, to establish a candidate transcription factor that may be involved in VCAF gene regulation, we investigated the effect of dexamethasone on the NF-B protein in pericytes. Immunocytochemical analysis confirmed significant NF-B staining in both the cytoplasmic compartment and to a lesser extent, in the nuclear compartment in control pericytes (Figure 7A, a and c). However, after dexamethasone treatment, there was an apparent reduction in nuclear staining of NF-B (Figure 7A, b and d). Western blot analysis of total protein lysates obtained from pericytes cultured with and without dexamethasone revealed a decrease in total levels of NF-B protein in the dexamethasone-treated versus control cells (Figure 7B). NF-B was restored to control levels when cells were incubated in the presence of dexamethasone plus Org 34116, indicating a transcriptional level of control of this gene via the GR (Figure 7B). Fractionation of the cells, followed by Western blot analysis, confirmed a change in the subcellular distribution of NF-B, whereby a pronounced decrease in NF-B in the nuclear compartment of pericytes treated with dexamethasone compared with controls was demonstrated (Figure 7C).
Discussion
This is the first report demonstrating that dexamethasone enhances the osteogenic differentiation of bovine retinal pericytes in vitro and investigating the mechanism by which this glucocorticoid exerts its effects. We demonstrate that dexamethasone treatment results in enhanced osteogenic differentiation of pericytes, an effect abolished in the presence of the GR antagonist Org 34116. Dexamethasone induced an upregulation of the osteoblast-specific marker ALP, increased nodule formation, and calcium deposition, as well as downregulated the postulated calcification-inhibitor genes MGP, OPN, and VCAF. Proliferation was unaffected by dexamethasone treatment of pericytes.
Previous studies show that vascular pericytes are capable of differentiating into osteoblast-like cells in vitro and in vivo.5 Bone formation has been shown to occur in the vasculature,19 and Bostrom et al propose a developmental retention of pluripotent cells coupled with a loss of molecular regulatory control that unmasks an embryonic osteogenic program when given an appropriate stimulus.20 We speculate that pericyte-like cells represent a potential source of osteoprogenitor cells in the adult and, under certain stress situations, have their normal signaling pathways deregulated. For example, under the microenvironmental conditions associated with atherosclerosis, eg, inflammation and elevated oxidative stress, these cells could adopt an osteogenic phenotype and deposit a calcified matrix in the vasculature.
Many reports have been made on the paradox of osteoporosis and vascular calcification occurring together in many patients,17,21 adding strength to the idea that vascular calcification is a cellular and genetic process, rather than nonspecific calcium precipitation. Although glucocorticoids are known to induce osteoporosis, it appears that in vitro, glucocorticoids promote recruitment, differentiation, and maturation of osteoblasts.12,13,22 In the present study, dexamethasone was used at a physiological concentration of 10 nmol/L, which routinely induces the differentiation of human bone marrow stromal cells in vitro.11,12 We demonstrate a significant increase in the number and size of nodules formed by pericytes, continuously cultured in the presence of dexamethasone, compared with control cells. It is important to note that cells isolated in the presence and absence of dexamethasone appeared identical in terms of their expression of phenotypic markers of pericytes.5,16 However, in accordance with previous data,22,23 we cannot exclude the possibility that dexamethasone may have increased the proportion of osteoprogenitor cells in the original cell isolates. Taken together, these findings suggest that pericyte cultures contain early progenitors that are more responsive to dexamethasone than those that have become committed to a specific differentiation lineage.22
Our results also demonstrate that dexamethasone has no direct effect per se on the proliferation of pericytes in culture. These data are consistent with those of Mori et al,14 who also have shown that dexamethasone has no effect on VSMC proliferation. This is in contrast to the finding that high doses of dexamethasone inhibit the proliferation of osteoblasts precursors and osteoblasts in vitro.12,24 The reason for the disparity in these results is unclear but could be attributed to variations in the doses of dexamethasone used12 or in the cell types, pericytes versus VSMCs versus osteoblasts.
ALP activity is usually associated with the induction of bone formation and is considered a marker of osteogenic differentiation.25 We demonstrate an increase in ALP activity in both vehicle- and dexamethasone-treated cells. Furthermore, ALP activity is significantly elevated when pericytes are cultured continuously in the presence of dexamethasone in vitro compared with controls. In addition, there is an elevation in calcium deposition during pericyte osteogenic differentiation, the effect being markedly enhanced in the presence of dexamethasone. Based on our findings that dexamethasone accelerates pericyte nodule formation and mineralization, we hypothesized that glucocorticoids may alter the balance of inducers or inhibitors of differentiation and mineralization in vascular pericytes. To test this hypothesis, using Northern blot analysis, we investigated the expression of MGP and OPN, proposed markers of osteoblastic differentiation5,26 and inhibitors of mineralization,27,28 together with another gene, VCAF, also proposed as a potential inhibitor of mineralization.18 Although the mRNA abundance of all 3 genes was elevated during 3 weeks of dexamethasone treatment, there was a significant reduction in MGP and OPN and, to a lesser extent, VCAF mRNA abundance in pericytes after treatment with dexamethasone compared with controls at all time points.
The dexamethasone-induced downregulation of OPN and VCAF in pericytes, together with the increased mineral deposition by these cells, lends support to the hypothesis that these proteins may act as inhibitors of mineralization.18,27 The effects of the downregulation of MGP expression by dexamethasone on the osteogenic differentiation and mineralization of pericytes is more complex because MGP has been shown to both (1) regulate the osteogenic differentiation of vascular cells by controlling the availability of bone morphogenetic protein 2 (BMP-2)26,29,30 and (2) inhibit mineralization in vivo.28 Therefore, in future studies, it would be interesting to determine whether BMP-2 expression is also modulated by dexamethasone and whether this protein might also be involved in the accelerated mineralization observed in dexamethasone-treated pericytes.
There are reports indicating that dexamethasone-mediated suppression of mRNA occurs at the posttranscriptional level as a result of mRNA destabilization.31 We investigated whether dexamethasone could downregulate MGP, OPN, and VCAF mRNA by increasing their mRNA degradation. However, when transcription was blocked by actinomycin D, there was no evidence for posttranscriptional control of gene expression, as the half-lives of all 3 mRNAs were more than 8 hours and unaffected by the presence of dexamethasone. These results eliminate destabilization of MGP, OPN, and VCAF mRNAs as a mechanism by which dexamethasone downregulates their expression during pericyte differentiation.
Endogenous systemic glucocorticoids modulate the expression of genes by activating the GR, which is a ligand-activated transcription factor of the nuclear-receptor family.32 The GR resides in the cytoplasm as a multiprotein complex, which, on ligand binding, dissociates and translocates into the nucleus to up- or downregulate mRNA synthesis.32 We used the GR antagonist Org 34116 and demonstrate that it (1) blocks the dexamethasone-induced increase in nodule formation and size, (2) blocks the dexamethasone-induced increase in calcium deposition, and (3) blocks the inhibitory effect of dexamethasone on MGP mRNA expression. These data strongly suggest that the dexamethasone–GR complex is a likely mediator not only of accelerated osteogenic differentiation and mineralization of pericytes but also of MGP gene repression, possibly by direct action of the GR on a negative glucocorticoid response element (GRE).32 In support of this suggestion, 2 potential negative GREs33 are present in the MGP promoter sequence.34 In contrast, cyclohexamide treatment had no effect on the dexamethasone-induced decrease of MGP, ruling out the involvement of another protein in the downregulation of MGP mRNA by dexamethasone and adding strength to a direct effect on a negative GRE.
OPN mRNA remained downregulated after dexamethasone plus Org 34116 treatment, eliminating transcriptional regulation as a mechanism of control by dexamethasone. This was a surprising result, because a GRE has been identified within the human osteopontin promoter.35 However, Wang et al have also suggested that under different conditions and within different cell types, the context of the GRE and other transcription factors could influence the binding of the GR to a promoter.35 Cyclohexamide also had no effect on dexamethasone-induced OPN downregulation, suggesting posttranslational control by the GR, perhaps involving a phosphorylation event via another protein. In this regard, it is recognized that phosphorylation of proteins is an important mechanism that controls cellular events in the nucleus.36 These data indicate that some form of alternative signaling and/or crosstalk between pathways is in play, adding support to the suggestion that the GR is present in a range of multiprotein complexes (receptosomes) with different functions and means of gene regulation.37 Clearly, a future study is warranted to understand the effects of dexamethasone on OPN gene regulation, because GR-interacting protein complexes may play a role in the variability of glucocorticoid responsiveness observed in some patients.
Org 34116 blocked the inhibitory effect of dexamethasone on VCAF mRNA and protein expression (see the online data supplement available at http://circres.ahajournals.org), which, like MGP, suggests involvement of the dexamethasone–GR complex possibly via a negative GRE. We also investigated the involvement of de novo protein synthesis in VCAF regulation by dexamethasone and found that, in contrast to MGP and OPN, cyclohexamide blocked dexamethasone-induced downregulation of VCAF mRNA. These results suggest a requirement for de novo protein synthesis as well, perhaps indicating protein–protein interaction with other sequence-specific transcription factors.
The most widespread advantage of glucocorticoids is their antiinflammatory effect, thought to be attributable to a direct inhibitory interaction between activated GRs and transcription factors, such as NF-B.38 Inactive NF-B is located in the cytoplasmic compartment of the cell in association with the I-B inhibitory protein. On stimulation by inflammatory agents, I-B dissociates from NF-B, allowing translocation of NF-B to the nucleus, where it acts as a transcription factor.39 We now demonstrate that NF-B is negatively controlled at the level of transcription by dexamethasone, resulting in less NF-B being translated into protein. In addition, we show, by immunocytochemistry, that dexamethasone treatment results in an apparent decrease in NF-B protein in the nucleus. Although immunofluorescence studies may be limited by the fact that cell shape changes can influence the amount of cytoplasm over the nucleus and hence the apparent amount of labeled NF-B within the nucleus, we confirm these results using Western blot analysis and demonstrate a reduced level of NF-B protein in the nuclear compartment after dexamethasone treatment. These results suggest that this transcription factor may be involved in the regulation of pericyte differentiation and mineralization by dexamethasone. Furthermore, we suggest that the reduction of NF-B in the nucleus could be responsible for reduced transcription of VCAF in pericytes after treatment with dexamethasone. However, further studies are required to verify the interaction and regulation of the GR multiprotein complex, its effects on calcification-inhibiting proteins such as VCAF, and the signaling pathways involved in their regulation.
In conclusion, our results support the proposal that the vasculature could have a population of cells that, in response to dexamethasone, differentiate toward an osteoblastic lineage by upregulating osteogenic markers and downregulating specific inhibitor molecules in both a direct and indirect manner. Our data identify downstream targets of glucocorticoid activation and suggest that elevated differentiation and mineralization of vascular pericytes by dexamethasone is associated with the loss of expression of mineralization inhibitory genes, which are controlled by dexamethasone at multiple levels. This study unravels a novel mechanism for transcriptional regulation by the GR and may be an important component in the cellular differentiation associated with vascular calcification. We speculate that glucocorticoids per se promote vascular calcification mostly by inhibiting calcification-inhibitor molecules as the main target, partly by decreasing NF-B transcription and activation. These data provide novel concepts regarding glucocorticoid-signaling pathways and vascular calcification. Identification of tissue-specific gene regulatory networks could yield insights into the molecular basis of vascular calcification and may lead to improved treatment regimes for diseases where glucocorticoid therapy is in use.
Acknowledgments
This study was supported in part by grant PG/02/120/14492 from the British Heart Foundation (to M.Y.A. and A.E.C.) and by a Biological and Biochemical Sciences Research Council studentship (to J.P.K.).
Footnotes
Original received November 17, 2005; revision received March 31, 2006; accepted April 6, 2006.
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the Wellcome Trust Centre for Cell-Matrix Research and Division of Cardiovascular and Endocrine Sciences, Faculty of Medical and Human Sciences, University of Manchester, UK.
Abstract
Vascular calcification is present in many pathological conditions and is recognized as a strong predictor of future cardiovascular events. Current evidence suggests that it is a regulated process involving inducing and inhibitory molecules. Glucocorticoids have great clinical importance as antiinflammatory drugs and can act as potent inducers of osteogenic differentiation in vitro. The effect of glucocorticoids on vascular cells in vivo remains obscure. Pericytes are pluripotent cells that can differentiate into osteoblasts, and recent evidence suggests that they could participate in vascular calcification. We hypothesized that the synthetic glucocorticoid dexamethasone would enhance the rate of pericyte differentiation and mineralization in vitro with a concomitant suppression of calcification-inhibitory molecules. Three weeks of dexamethasone treatment induced a 2-fold increase in (1) alkaline phosphatase activity, (2) calcium deposition, and (3) the number of nodules formed in vitro; and a reduction in the expression of matrix Gla protein (MGP), osteopontin (OPN), and vascular calcification-associated factor (VCAF) mRNAs. The glucocorticoid receptor antagonist Org 34116 abolished dexamethasone-accelerated pericyte differentiation, nodule formation, and mineralization. Data obtained using Org 34116, the transcription inhibitor actinomycin D, and the protein synthesis inhibitor cyclohexamide suggest that MGP, OPN, and VCAF mRNA abundance are controlled at different and multiple levels by dexamethasone. This is the first report showing that dexamethasone enhances the osteogenic differentiation of pericytes and downregulates genes associated with inhibition of mineralization. Our study highlights the need for further investigation into the long-term consequences of prolonged glucocorticoid therapy on vascular calcification.
Key Words: pericytes differentiation calcification gene regulation glucocorticoid
Introduction
Calcification can lead to devastating clinical consequences when present in blood vessels. It has been suggested that cells maintain a balance between calcification inducing and inhibitory proteins in soft tissues in such a way that the deposition of a mineralized matrix is normally avoided. Alterations in this balance may be brought about by injury or disease, with the subsequent deposition of calcific deposits in the vessel wall. It is now well established that vascular calcification is associated with elevated expression of bone-related proteins1,2 and that pericyte-like cells exist in the vasculature, which, under appropriate conditions, can be induced to display characteristics of the osteoblastic lineage.3,4 Our previous studies have demonstrated that cultured postconfluent pericytes form large calcified multicellular nodules both in vitro and in vivo,5 the matrix of which resembles that found in calcified atherosclerotic plaques and bone.5 Although factors such as transforming growth factor (TGF-),6 tumor necrosis factor (TNF-),7 and acetylated LDL8 have been shown to be involved in the promotion of calcification in vitro, the underlying mechanisms of vascular calcification have not been fully elucidated.
The main risk factors for atherosclerosis include not only the classic factors such as age, high cholesterol levels, and hypertension but also prolonged glucocorticoid therapy. The mechanisms underlying the adverse effects of glucocorticoid excess on the vascular system are complex and have not yet been fully explained. It is well recognized that vascular ossification and osteoporosis often coexist in patients, whereby bone mineral is formed within the vasculature, while it is simultaneously lost from the skeleton.9 How this buildup and loss of mineral can occur simultaneously in patients remains to be elucidated.
Glucocorticoids are a class of steroid hormones produced by the cortex within the mammalian adrenal gland and are normally maintained at steady-state levels. A deregulation of endogenous levels is induced by vascular injury in vivo10 and could be a contributing factor in the development of vascular calcification. The synthetic glucocorticoid dexamethasone induces an osteoblastic differentiation pathway in many different cell types in vitro, including human bone marrow–derived stromal cells,11,12 fetal rat calvarial cells,13 bovine vascular smooth muscle cells (SMCs),14 and mouse embryo–derived NIH3T3 fibroblasts.15 This study tests the hypothesis that dexamethasone enhances the in vitro osteoblastic differentiation of vascular pericytes and that this may be associated with a decrease in the expression of calcification-inhibitor molecules. We investigate several parameters within pericytes exposed to dexamethasone, in terms of their proliferation, the time-course of nodule formation and mineralization, the expression of osteoblastic markers, and the mechanisms underlying these effects.
Materials and Methods
Cell Culture and Nodule Formation
Pericytes were isolated from bovine retinas essentially as previously described,16 except that cells were isolated and subsequently maintained either in the continued presence or absence of dexamethasone (10 nmol/L) in Eagle’s minimal essential medium (MEM) containing 20% FCS, 2 mmol/L glutamine, 1 mmol/L sodium pyruvate, 50 μg/mL ascorbate-2-phosphate, and nonessential amino acids. Control cells were treated with vehicle alone. For experiments, pericytes were plated at 1.5x104 cells/cm2, with a media change every 4 days. In some experiments, the glucocorticoid receptor (GR) antagonist Org 34116 (1 μmol/L; a kind gift from Angela Fisher, Organon, Motherwell, Scotland, UK) was also included, and the cells were subsequently harvested at weekly time points. The area occupied by nodules was determined using AnalySIS software. Mineralized nodules were visualized by washing in PBS, fixing in 4% formaldehyde, and staining with 1% (wt/vol) Alizarin red S (pH 4.2). Actinomycin D and cyclohexamide were used at a final concentration of 5 μg/mL and 35 mmol/L, respectively. Cells were grown for 3 weeks to ensure detectable baseline levels of vascular calcification-associated factor (VCAF), osteopontin (OPN), and matrix Gla protein (MGP) mRNA and protein; then actinomycin D or cyclohexamide was added to the media for 8 and 4 hours, respectively, before harvesting.
Alkaline Phosphatase Activity
Cells were seeded in 6-well plates and maintained up to 4 weeks. Proteins were extracted at weekly time points by freeze-thawing the cells in 0.05% Triton X-100 in PBS. Total cellular proteins were measured using Bradford’s assay (Bio-Rad). Alkaline phosphatase (ALP) activity was quantified in protein lysates (15 μg) made up to 100 μL in 2-amino-2-methyl 1-propanol (AMP) buffer containing 8 mmol/L -nitrophenylphosphate (-NPP) (Sigma-Aldrich) as substrate and incubated at 37°C for 2 hours before terminating the reaction with 0.5 mol/L NaOH (100 μL). The absorbance was measured at 405 nm and ALP activity calculated as nmol/L -nitrophenol converted per microgram of protein per minute.
Calcium Assay
45Ca deposition was assayed as described.17 Cells were seeded in 24-well plates and harvested weekly up to 4 weeks. 45CaCl2 (ICN) was added (1.0 μCi/mL for 48 hours), and the cells were harvested in PBS (0.3 mL), placed into vials containing perchloric acid (0.2 mL) with 3% hydrogen peroxide (0.3 mL), and incubated at 80°C for 1 hour. Samples were then dissolved in ethylene glycol monoethyl ether (0.6 mL), and the radioactivity was quantified by scintillation counting.
RNA Analysis
Cells were seeded in 25-cm2 flasks and the RNA harvested weekly using TRIzol reagent (Invitrogen). Northern blot analysis and probe preparation were performed as previously described.5,18 Densitometry was used to express the mRNA abundance of MGP, OPN, and VCAF relative to 18S ribosomal RNA. To determine mRNA stability, total RNA was harvested at selected time points following incubation with the transcriptional inhibitor actinomycin D. mRNA abundance at the time of addition of actinomycin D was set at 100%, and the rate of OPN, MGP, and VCAF mRNA decay was compared in dexamethasone- versus vehicle-treated cells.
Western Blot and Immunocytochemical Analysis
Whole cell lysates were extracted as previously described,16 and cytosolic and nuclear fractions were separated using a BioVision kit (Mountain View, Calif) according to the protocol of the manufacturer. Proteins (20 μg/lane) were separated by SDS-PAGE (10% gels) and transferred to nitrocellulose membranes (Amersham Biosciences). The membranes were incubated with a nuclear factor B (NF-B) antibody (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, Calif) at 4°C for 16 hours and a horseradish peroxidase–conjugated swine anti-rabbit secondary antibody (1:1000 dilution) and developed as described.16 For immunocytochemistry, pericytes were cultured in chamber slides for 48 hours, fixed in 100% methanol, and blocked in 5% goat serum/PBS for 1 hour at room temperature. The NF-B polyclonal antibody (1:200 dilution) was used with a goat anti-rabbit IgG, conjugated to AlexaFluor 488 secondary antibody (Molecular Probes; 1:300 dilution). Cells were counterstained with 4 μg/mL 4',6-diamidino-2-phenylindole (DAPI).
Data Analysis
The data for the ALP, nodule formation, and 45Ca assay are shown as the mean±SD of 2 experiments performed in triplicate wells. An unpaired, 2-tailed Student’s t test or ANOVA was used to determine statistical significance between mean values, as appropriate. A value of P<0.05 was considered statistically significant. For Northern and Western blots, data are representative of at least 2 experiments.
Results
Dexamethasone Induces Differentiation and Mineralization of Pericytes In Vitro
Initially, the effects of continued presence or absence of dexamethasone on pericyte proliferation, alkaline phosphatase activity, calcium incorporation, and mineralized nodule formation were investigated. Using a thymidine incorporation assay, pericyte growth was investigated during the first week of culture, and dexamethasone was found to have no significant effect on pericyte proliferation compared with controls (data not shown).
There was a 4.4-fold increase in ALP activity in pericytes after dexamethasone treatment during 3 weeks of culture compared with 2.1-fold increase in the vehicle-treated cells (Figure 1). At week 3, there was a 2.2-fold increase in ALP activity in cells grown in the presence of dexamethasone compared with vehicle-treated cells (Figure 1). No further increase in ALP activity was detected at week 4.
Nodule formation was detected as early as 1 week after plating in the presence of dexamethasone (Figure 2A), culminating in a 2.9-fold increase in the number of nodules formed, compared with vehicle-treated cells at week 4 (Figure 2B). In addition, nodules were larger (Table) and mineralized earlier in the presence of dexamethasone compared with controls (Figure 2A). Furthermore, in the presence of the glucocorticoid antagonist Org 34116, the dexamethasone-induced increase in pericyte nodule number and size was abolished (Table).
Culturing pericytes for 4 weeks in dexamethasone increased 45Ca deposition 2.4-fold compared with vehicle-treated cells (Figure 3). The increased calcium incorporation in the dexamethasone-treated cells was also abolished in the presence of Org 34116 (Figure 3).
Dexamethasone Reduces OPN, MGP, and VCAF mRNA Abundance in Pericytes
MGP, OPN, and VCAF mRNAs were examined in cells maintained in the presence and absence of dexamethasone. As reported previously, we detected an increase in expression of OPN,5 MGP, and VCAF mRNA18 in vehicle-treated control cells with increasing time in culture (Figure 4). All 3 genes were also upregulated in pericytes cultured in the presence of dexamethasone for 2 to 3 weeks (Figure 4); however, dexamethasone caused a decrease in the mRNA abundance of all 3 transcripts compared with vehicle-treated cells (Figure 4), with the decrease in MGP and OPN mRNA being particularly striking (Figure 4B and 4C). These differences in gene expression were maintained when pericytes were cultured with and without dexamethasone for 4 weeks (results not shown).
Control of MGP, OPN, and VCAF Gene Expression by Dexamethasone
The decline in abundance of MGP, OPN, and VCAF mRNAs could be caused directly by either a suppression of transcription or accelerated degradation of RNA or indirectly by the involvement of a second transcription factor. mRNA stability was investigated using the transcription inhibitor actinomycin D. MGP, OPN, and VCAF mRNAs were found to have long half-lives of more than 8 hours, which were not altered by culturing cells in the presence of dexamethasone (data not shown), suggesting that dexamethasone had not induced degradation of these mRNAs.
To investigate transcriptional control mediated by dexamethasone binding to its receptor, the cells were treated with Org 34116. Pericytes were divided into 3 groups: (1) without dexamethasone (–), (2) with dexamethasone (+), and (3) in the presence of dexamethasone plus Org 34116. The presence of Org 34116 blocked the dexamethasone-induced downregulation of MGP and VCAF mRNAs (Figure 5A, 5B, and 5D), adding support for a transcriptional mechanism of regulation of these genes by dexamethasone. However, the GR antagonist had no effect on OPN expression after dexamethasone treatment (Figure 5A and 5C), indicating that dexamethasone regulates OPN gene expression by a different mechanism to that shown for MGP and VCAF.
Because OPN expression was unaffected by the GR antagonist, it is likely that another protein is involved in the regulation of OPN by dexamethasone. To investigate this possibility and to assess the role of de novo protein synthesis in the downregulation of MGP, VCAF, and OPN mRNA during continuous exposure to dexamethasone, pericytes were treated with the protein synthesis inhibitor cyclohexamide for 4 hours (Figure 6). As previously shown, dexamethasone downregulated expression of MGP and OPN mRNA compared with control cells. However, the mRNA abundance of both MGP and OPN was found to be unaffected by cyclohexamide treatment (Figure 6). In contrast, cyclohexamide abolished the modest downregulation of VCAF mRNA expression after exposure of the cells to dexamethasone, suggesting involvement of another protein in VCAF regulation by dexamethasone (Figure 6).
Dexamethasone Prevents Nuclear Accumulation of NF-B in Pericytes
Growing evidence suggests that the antiinflammatory effects of glucocorticoids are mediated by a downregulation of NF-B. Therefore, to establish a candidate transcription factor that may be involved in VCAF gene regulation, we investigated the effect of dexamethasone on the NF-B protein in pericytes. Immunocytochemical analysis confirmed significant NF-B staining in both the cytoplasmic compartment and to a lesser extent, in the nuclear compartment in control pericytes (Figure 7A, a and c). However, after dexamethasone treatment, there was an apparent reduction in nuclear staining of NF-B (Figure 7A, b and d). Western blot analysis of total protein lysates obtained from pericytes cultured with and without dexamethasone revealed a decrease in total levels of NF-B protein in the dexamethasone-treated versus control cells (Figure 7B). NF-B was restored to control levels when cells were incubated in the presence of dexamethasone plus Org 34116, indicating a transcriptional level of control of this gene via the GR (Figure 7B). Fractionation of the cells, followed by Western blot analysis, confirmed a change in the subcellular distribution of NF-B, whereby a pronounced decrease in NF-B in the nuclear compartment of pericytes treated with dexamethasone compared with controls was demonstrated (Figure 7C).
Discussion
This is the first report demonstrating that dexamethasone enhances the osteogenic differentiation of bovine retinal pericytes in vitro and investigating the mechanism by which this glucocorticoid exerts its effects. We demonstrate that dexamethasone treatment results in enhanced osteogenic differentiation of pericytes, an effect abolished in the presence of the GR antagonist Org 34116. Dexamethasone induced an upregulation of the osteoblast-specific marker ALP, increased nodule formation, and calcium deposition, as well as downregulated the postulated calcification-inhibitor genes MGP, OPN, and VCAF. Proliferation was unaffected by dexamethasone treatment of pericytes.
Previous studies show that vascular pericytes are capable of differentiating into osteoblast-like cells in vitro and in vivo.5 Bone formation has been shown to occur in the vasculature,19 and Bostrom et al propose a developmental retention of pluripotent cells coupled with a loss of molecular regulatory control that unmasks an embryonic osteogenic program when given an appropriate stimulus.20 We speculate that pericyte-like cells represent a potential source of osteoprogenitor cells in the adult and, under certain stress situations, have their normal signaling pathways deregulated. For example, under the microenvironmental conditions associated with atherosclerosis, eg, inflammation and elevated oxidative stress, these cells could adopt an osteogenic phenotype and deposit a calcified matrix in the vasculature.
Many reports have been made on the paradox of osteoporosis and vascular calcification occurring together in many patients,17,21 adding strength to the idea that vascular calcification is a cellular and genetic process, rather than nonspecific calcium precipitation. Although glucocorticoids are known to induce osteoporosis, it appears that in vitro, glucocorticoids promote recruitment, differentiation, and maturation of osteoblasts.12,13,22 In the present study, dexamethasone was used at a physiological concentration of 10 nmol/L, which routinely induces the differentiation of human bone marrow stromal cells in vitro.11,12 We demonstrate a significant increase in the number and size of nodules formed by pericytes, continuously cultured in the presence of dexamethasone, compared with control cells. It is important to note that cells isolated in the presence and absence of dexamethasone appeared identical in terms of their expression of phenotypic markers of pericytes.5,16 However, in accordance with previous data,22,23 we cannot exclude the possibility that dexamethasone may have increased the proportion of osteoprogenitor cells in the original cell isolates. Taken together, these findings suggest that pericyte cultures contain early progenitors that are more responsive to dexamethasone than those that have become committed to a specific differentiation lineage.22
Our results also demonstrate that dexamethasone has no direct effect per se on the proliferation of pericytes in culture. These data are consistent with those of Mori et al,14 who also have shown that dexamethasone has no effect on VSMC proliferation. This is in contrast to the finding that high doses of dexamethasone inhibit the proliferation of osteoblasts precursors and osteoblasts in vitro.12,24 The reason for the disparity in these results is unclear but could be attributed to variations in the doses of dexamethasone used12 or in the cell types, pericytes versus VSMCs versus osteoblasts.
ALP activity is usually associated with the induction of bone formation and is considered a marker of osteogenic differentiation.25 We demonstrate an increase in ALP activity in both vehicle- and dexamethasone-treated cells. Furthermore, ALP activity is significantly elevated when pericytes are cultured continuously in the presence of dexamethasone in vitro compared with controls. In addition, there is an elevation in calcium deposition during pericyte osteogenic differentiation, the effect being markedly enhanced in the presence of dexamethasone. Based on our findings that dexamethasone accelerates pericyte nodule formation and mineralization, we hypothesized that glucocorticoids may alter the balance of inducers or inhibitors of differentiation and mineralization in vascular pericytes. To test this hypothesis, using Northern blot analysis, we investigated the expression of MGP and OPN, proposed markers of osteoblastic differentiation5,26 and inhibitors of mineralization,27,28 together with another gene, VCAF, also proposed as a potential inhibitor of mineralization.18 Although the mRNA abundance of all 3 genes was elevated during 3 weeks of dexamethasone treatment, there was a significant reduction in MGP and OPN and, to a lesser extent, VCAF mRNA abundance in pericytes after treatment with dexamethasone compared with controls at all time points.
The dexamethasone-induced downregulation of OPN and VCAF in pericytes, together with the increased mineral deposition by these cells, lends support to the hypothesis that these proteins may act as inhibitors of mineralization.18,27 The effects of the downregulation of MGP expression by dexamethasone on the osteogenic differentiation and mineralization of pericytes is more complex because MGP has been shown to both (1) regulate the osteogenic differentiation of vascular cells by controlling the availability of bone morphogenetic protein 2 (BMP-2)26,29,30 and (2) inhibit mineralization in vivo.28 Therefore, in future studies, it would be interesting to determine whether BMP-2 expression is also modulated by dexamethasone and whether this protein might also be involved in the accelerated mineralization observed in dexamethasone-treated pericytes.
There are reports indicating that dexamethasone-mediated suppression of mRNA occurs at the posttranscriptional level as a result of mRNA destabilization.31 We investigated whether dexamethasone could downregulate MGP, OPN, and VCAF mRNA by increasing their mRNA degradation. However, when transcription was blocked by actinomycin D, there was no evidence for posttranscriptional control of gene expression, as the half-lives of all 3 mRNAs were more than 8 hours and unaffected by the presence of dexamethasone. These results eliminate destabilization of MGP, OPN, and VCAF mRNAs as a mechanism by which dexamethasone downregulates their expression during pericyte differentiation.
Endogenous systemic glucocorticoids modulate the expression of genes by activating the GR, which is a ligand-activated transcription factor of the nuclear-receptor family.32 The GR resides in the cytoplasm as a multiprotein complex, which, on ligand binding, dissociates and translocates into the nucleus to up- or downregulate mRNA synthesis.32 We used the GR antagonist Org 34116 and demonstrate that it (1) blocks the dexamethasone-induced increase in nodule formation and size, (2) blocks the dexamethasone-induced increase in calcium deposition, and (3) blocks the inhibitory effect of dexamethasone on MGP mRNA expression. These data strongly suggest that the dexamethasone–GR complex is a likely mediator not only of accelerated osteogenic differentiation and mineralization of pericytes but also of MGP gene repression, possibly by direct action of the GR on a negative glucocorticoid response element (GRE).32 In support of this suggestion, 2 potential negative GREs33 are present in the MGP promoter sequence.34 In contrast, cyclohexamide treatment had no effect on the dexamethasone-induced decrease of MGP, ruling out the involvement of another protein in the downregulation of MGP mRNA by dexamethasone and adding strength to a direct effect on a negative GRE.
OPN mRNA remained downregulated after dexamethasone plus Org 34116 treatment, eliminating transcriptional regulation as a mechanism of control by dexamethasone. This was a surprising result, because a GRE has been identified within the human osteopontin promoter.35 However, Wang et al have also suggested that under different conditions and within different cell types, the context of the GRE and other transcription factors could influence the binding of the GR to a promoter.35 Cyclohexamide also had no effect on dexamethasone-induced OPN downregulation, suggesting posttranslational control by the GR, perhaps involving a phosphorylation event via another protein. In this regard, it is recognized that phosphorylation of proteins is an important mechanism that controls cellular events in the nucleus.36 These data indicate that some form of alternative signaling and/or crosstalk between pathways is in play, adding support to the suggestion that the GR is present in a range of multiprotein complexes (receptosomes) with different functions and means of gene regulation.37 Clearly, a future study is warranted to understand the effects of dexamethasone on OPN gene regulation, because GR-interacting protein complexes may play a role in the variability of glucocorticoid responsiveness observed in some patients.
Org 34116 blocked the inhibitory effect of dexamethasone on VCAF mRNA and protein expression (see the online data supplement available at http://circres.ahajournals.org), which, like MGP, suggests involvement of the dexamethasone–GR complex possibly via a negative GRE. We also investigated the involvement of de novo protein synthesis in VCAF regulation by dexamethasone and found that, in contrast to MGP and OPN, cyclohexamide blocked dexamethasone-induced downregulation of VCAF mRNA. These results suggest a requirement for de novo protein synthesis as well, perhaps indicating protein–protein interaction with other sequence-specific transcription factors.
The most widespread advantage of glucocorticoids is their antiinflammatory effect, thought to be attributable to a direct inhibitory interaction between activated GRs and transcription factors, such as NF-B.38 Inactive NF-B is located in the cytoplasmic compartment of the cell in association with the I-B inhibitory protein. On stimulation by inflammatory agents, I-B dissociates from NF-B, allowing translocation of NF-B to the nucleus, where it acts as a transcription factor.39 We now demonstrate that NF-B is negatively controlled at the level of transcription by dexamethasone, resulting in less NF-B being translated into protein. In addition, we show, by immunocytochemistry, that dexamethasone treatment results in an apparent decrease in NF-B protein in the nucleus. Although immunofluorescence studies may be limited by the fact that cell shape changes can influence the amount of cytoplasm over the nucleus and hence the apparent amount of labeled NF-B within the nucleus, we confirm these results using Western blot analysis and demonstrate a reduced level of NF-B protein in the nuclear compartment after dexamethasone treatment. These results suggest that this transcription factor may be involved in the regulation of pericyte differentiation and mineralization by dexamethasone. Furthermore, we suggest that the reduction of NF-B in the nucleus could be responsible for reduced transcription of VCAF in pericytes after treatment with dexamethasone. However, further studies are required to verify the interaction and regulation of the GR multiprotein complex, its effects on calcification-inhibiting proteins such as VCAF, and the signaling pathways involved in their regulation.
In conclusion, our results support the proposal that the vasculature could have a population of cells that, in response to dexamethasone, differentiate toward an osteoblastic lineage by upregulating osteogenic markers and downregulating specific inhibitor molecules in both a direct and indirect manner. Our data identify downstream targets of glucocorticoid activation and suggest that elevated differentiation and mineralization of vascular pericytes by dexamethasone is associated with the loss of expression of mineralization inhibitory genes, which are controlled by dexamethasone at multiple levels. This study unravels a novel mechanism for transcriptional regulation by the GR and may be an important component in the cellular differentiation associated with vascular calcification. We speculate that glucocorticoids per se promote vascular calcification mostly by inhibiting calcification-inhibitor molecules as the main target, partly by decreasing NF-B transcription and activation. These data provide novel concepts regarding glucocorticoid-signaling pathways and vascular calcification. Identification of tissue-specific gene regulatory networks could yield insights into the molecular basis of vascular calcification and may lead to improved treatment regimes for diseases where glucocorticoid therapy is in use.
Acknowledgments
This study was supported in part by grant PG/02/120/14492 from the British Heart Foundation (to M.Y.A. and A.E.C.) and by a Biological and Biochemical Sciences Research Council studentship (to J.P.K.).
Footnotes
Original received November 17, 2005; revision received March 31, 2006; accepted April 6, 2006.
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