all-trans-Retinoic Acid Increases Nitric Oxide Synthesis by Endothelial Cells
A Role for the Induction of Dimethylarginine Dimethylaminohydrolase
all-trans-Retinoic acid (atRA) has important effects on the developing and mature cardiovascular system. Nitric oxide (NO) production has been associated with the atRA-induced differentiation of neuronal cells, and we hypothesized that NO may also mediate certain actions of atRA in the cardiovascular system. We studied the effects of atRA on NO production by endothelial cells and determined whether regulation of enzymes responsible for metabolism of asymmetric dimethylarginine (ADMA) contributed to the effects seen. Murine endothelioma (sEnd.1) cells were incubated with or without atRA. Nitrite production was determined using the Griess reaction. The expression of NO synthase (NOS) and dimethylarginine dimethylaminohydrolase (DDAH) genes was determined by Northern blotting. A reporter gene assay was also used to study the effect of atRA on the DDAH II promoter. atRA significantly increased nitrite production by sEnd.1 cells despite no increase in eNOS expression. atRA also increased DDAH II gene expression and promoter activity and reduced the ratio of ADMA to symmetric dimethylarginine (SDMA) in culture medium. The DDAH inhibitor 4124W significantly reduced the induction of NO synthesis by atRA. The present study demonstrates that atRA increases NO synthesis in endothelial cells without increasing eNOS expression. atRA also increases the expression of DDAH II, the predominant DDAH isoform in endothelial cells. Our data suggests that the induction of NO synthesis by atRA may be facilitated by DDAH II. This pathway may help to explain some of the effects of atRA on the cardiovascular system.
- asymmetric methylarginine
- dimethylarginine dimethylaminohydrolase
- nitric oxide
- retinoic acid
Vitamin A derivatives (retinoids), and in particular all-trans-retinoic acid (atRA), inhibit cellular proliferation and promote cellular differentiation. These actions have important effects on the developing as well as the mature cardiovascular system and have potential therapeutic value. atRA regulates angiogenesis,1 may retard the development of atherosclerosis,2 inhibits neointima formation, and alters remodeling after vascular injury or bypass grafting.3–7⇓⇓⇓⇓ The mechanisms involved are poorly understood and attention has previously focused on vascular smooth muscle cell (VSMC) effects.8 However, there are good reasons to suspect that atRA may also affect the endothelium. Endothelial cells are exposed to the highest concentration of circulating atRA, express retinoid receptors, and play a significant role in atRA metabolism compared with other cell types.9 atRA also modulates endothelial cell growth, differentiation, and morphology.10,11⇓ Recent data suggests that the effects of retinol (vitamin A alcohol) on VSMC function may depend on an unknown endothelial factor.12
NO released by endothelial cells is an important regulator of vascular function. The cellular effects of NO bear some similarity to those of atRA, and NO has been implicated in the atRA-induced differentiation of neuronal cells.13 We therefore speculated that atRA might regulate NO production by endothelial cells. With respect to the possible mechanisms involved, we focused on the role of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase (NOS).14 ADMA has been described as an inhibitor of angiogenesis15 and a circulating marker of endothelial dysfunction16,17⇓ and is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). We and others have suggested that regulation of DDAH activity may be a mechanism to regulate NO production.18,19⇓
Materials and Methods
Cell Culture and Treatment
Five cell types were used. Murine endothelioma cells (sEnd.1)18 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS). sEnd.1 cells stably expressing a DDAH II promoter/reporter plasmid (sEnd.1D2A) were created by cotransfection of the reporter plasmid (pGL3sal) and a plasmid encoding a puromycin resistance gene (pBabe) at a ratio of 1:9 into sEnd.1 cells. sEnd.1D2A cells were cultured in DMEM containing 5% FCS. ECV304 cells (American Type Culture Collection, Ma- nassas, Va) were cultured in M199 medium containing 10% FCS. Primary porcine aortic endothelial cells (PAECs; single donor; passage 2 to 7) were prepared by the collagenase treatment of porcine aorta and cultured in DMEM containing 10% FCS. SV40 transfected human umbilical vein endothelial cells (SGHEC-7)18 were cultured in medium M199:RPMI 1640 in a ratio of 1:1 supplemented with 2.5 μg/mL endothelial cell growth supplement, 0.09 mg/mL heparin, 2.5% FCS, and 2.5% newborn calf serum. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and were routinely passaged every 7 days using trypsin/EDTA. atRA and 9-cisRA (Sigma) stock solutions were made up in DMSO and added to the medium to achieve a final concentration of 1 μmol/L for sEnd.1, PAECs, and SGHEC-7 cells. Concentrations of 10 μmol/L were used for promoter/reporter studies in ECV304 and sEnd.1D2A cells because this produced maximal upregulation of the reporter gene. Control cells were treated with DMSO only. Final DMSO concentrations in culture medium did not exceed 0.1% (v/v). In some experiments, the DDAH inhibitor, S-2-amino-4(3-methylguanidino) butanoic acid (4124W),18 ADMA, or symmetric dimethylarginine (SDMA) were added to the culture medium (final concentration 1 mmol/L). In other experiments, cells were exposed to actinomycin D (1.0 μg/mL) or cycloheximide (0.5 μg/mL) to block transcription or protein synthesis respectively. NO production by sEnd.1 cells was determined by measuring nitrite accumulation in cell culture medium (Griess reaction). Methylarginine concentrations in conditioned medium were determined by high-performance liquid chromatography (HPLC) as previously described.14
Total RNA was isolated from confluent cell monolayers grown in 6-well tissue culture plates (approximately 106 cells/well at the time of treatment) using TRIzol (Gibco-BRL) according to the manufacturer’s instructions. RNA samples were subjected to electrophoresis in 1% denaturing agarose gels, transferred to Hybond-N+ membranes (Amersham) according to the manufacturer’s instructions, and crosslinked using ultraviolet light. A murine DDAH I probe was obtained from an IMAGE clone (No. 732946, accession No. AA403666), which contains an insert identical to base pairs 371 to 870 of mouse DDAH I cDNA. A probe for mouse DDAH II was produced by RT-PCR of sEnd.1 cell total RNA using oligonucleotides mDDAH II.1 and mDDAH II.2 (complementary to base pairs −224 to −205 and 346 to 324 of mouse DDAH II cDNA, respectively). A probe for mouse eNOS was produced in a similar way using oligonucleotides mENOS1 and mENOS2 (complementary to base pairs 3310 to 3328 and 3621 to 3602 of mouse eNOS cDNA, respectively). Probes were labeled with 32P dCTP using a random primed labeling kit (Boehringer), purified on Nick columns (Pharmacia), and hybridized to the membranes according to the manufacturer’s instructions. Signals were visualized using a PhosphorImager (Fuji BAS1000 PhosphorImager) and quantified using TINA 2.08e software. Hybridization with β-actin cDNA was used to control for the amount of RNA isolated and loaded.
sEnd.1 cells were grown to confluence in 75-cm2 flasks and lysed with 0.1% SDS. Protein concentrations were determined using a Bio-Rad protein assay kit with bovine serum albumin standards. Protein samples (100 μg) were then separated on a 12% SDS-polyacrylamide gel under reducing conditions, transferred to a nitrocellulose membrane, and probed with a polyclonal antibody (Ab) specific for DDAH II. The DDAH II polyclonal Ab was raised in rabbit against a DDAH II241–255 peptide. Immunoreactive bands were visualized with a horseradish peroxidase conjugated secondary Ab using an ECL detection kit (Amersham).
Cloning and Sequencing of DDAH II Promoter Region
We identified a human genomic clone that contains the entire DDAH II gene.20 To study transcriptional regulation, we subcloned and sequenced 1.6 kb of the DNA immediately upstream of the DDAH II open reading frame. Sequence was analyzed for the presence of known transcription factor binding sites.
Reporter Gene Assay
A restriction fragment spanning nucleotides −1755 to −216 of the human DDAH II gene was isolated from a human DDAH II genomic clone and cloned into the multiple cloning site of the pGL3basic luciferase vector (Promega) to produce a DDAH II promoter/reporter gene construct (pGL3sal). Sequences containing the putative PPAR/RXR binding site were deleted by PCR. Oligonucleotides were designed to amplify −927 to −216 downstream of PPAR/RXR consensus site. This PCR product was cloned into pGL3 basic to produce pGL3salΔPPAR and sequenced. Other putative transcription binding sites were sequentially deleted in a series of promoter/reporter deletion constructs produced by cloning PCR generated fragments into pGL3basic. Transient transfections of ECV304 cells with promoter/reporter constructs (along with a β-galactosidase expression vector to control for transfection efficiency) were achieved using TransFast transfection reagent (Promega). ECV304 cells were used because they transfect at a higher efficiency than sEnd.1 cells using this method. Twenty-four hours after transfection, 10 μmol/L atRA was added to the culture medium and cells maintained in culture for a further 24 hours. Luciferase and β-galactosidase activity were measured using the Dual-Light chemiluminescent reporter gene assay system (Tropix Inc) according to manufacturer’s instructions. Results are expressed as luciferase activity normalized for β-galactosidase expression.
Data are expressed as mean±SEM values unless otherwise stated. A 2-way ANOVA test was used to analyze data from time-course experiments. Other statistical analyses were performed using the Student’s 2-tailed t test. Values of P<0.05 were regarded as being statistically significant.
Effect of atRA on NO Synthesis and NOS Expression
Nitrite production by sEnd.1 cells increased from 9.9±0.6 to 13.7±0.8 nmol/106 cells over 24 hours after stimulation by atRA (1 μmol/L) (n=9; P<0.005) (Figure 1A). No significant increase in nitrite generation was seen at 4, 8 (data not shown), or 12 hours. Nitrite was undetectable in media from stimulated and control cells treated with the NOS inhibitor ADMA (n=4), confirming that nitrite arose from NOS activity. The inactive enantiomer of ADMA, SDMA, did not affect nitrite accumulation (data not shown).
To determine whether the time-dependent changes in nitrite production might be associated with changes in NOS expression after atRA treatment, we studied NOS mRNA expression in sEnd.1 cells by Northern blotting. eNOS mRNA levels decreased by 5.8% and 20.3% at 12 and 24 hours, respectively (Figure 1B). Expression of nNOS or iNOS mRNA was not detectable at any time point (data not shown).
Effect of RA on DDAH Expression
Low levels of DDAH I mRNA were detectable in sEnd.1 cells and no significant changes in expression were seen between control and atRA-treated cells over the time course studied. In contrast, DDAH II mRNA levels in sEnd.1 cells increased in a time-dependent manner after atRA treatment (by 90% and 73% compared with controls at 12 and 24 hours, respectively) (Figure 1B). Western blotting confirmed that DDAH II protein expression was also increased by atRA treatment of sEnd.1 cells (Figure 1B). A similar effect was also seen in the human endothelial cell line SGHEC-7 (data not shown). To test whether atRA also induced DDAH II expression in primary arterial endothelial cells, we used PAECs. In primary PAECs, atRA increased DDAH II mRNA expression by 26.3±7.6% (P<0.01; n=6) 24 hours after treatment.
Effect of DDAH Regulation on Methylarginine Production
atRA (10 μmol/L) treatment of sEnd.1D2A cells for 48 hours caused a substantial and significant decrease in the ADMA/SDMA ratio in conditioned medium, characteristic of increased DDAH activity (control=14.14±1.9 versus atRA-treated=9.63±0.8; P<0.005, n=16 from 3 independent experiments) (Figure 2). The absolute concentration of SDMA increased, suggesting increased protein turnover in atRA-treated cells.
Effect of DDAH Regulation on NO Synthesis
To study whether or not the induction of DDAH II might contribute to the increase in nitrite production after atRA treatment, we used the DDAH inhibitor 4124W, which we have previously characterized and shown to alter vascular reactivity in organ bath studies.18 In this experiment, 4124W reduced nitrite release from control cells by a small but statistically insignificant amount (n=4). In contrast, 4124W reduced the atRA-stimulated increase in nitrite production by 53.5±9.2% (n=4; P<0.02) (Figure 3).
Effect of atRA on DDAH II Promoter Activity
atRA treatment of ECV304 cells increased DDAH II mRNA expression by 45.1±11.9% after 24 hours (n=3; P<0.02). This effect of atRA was due to transcriptional regulation as it was blocked by actinomycin D (Figure 4). atRA treatment of ECV304 cells transiently transfected with a human DDAH II promoter/reporter construct increased promoter activity by 46.6±7.4% (n=4; P<0.05; Figure 5A) compared with controls. Analysis of the promoter region identified consensus binding sites for several transcription factors, including a PPAR/RXR site (Figure 5B). This site is the only candidate site for a direct effect of retinoids. The natural ligand for RXR is 9cisRA and in many cell types atRA can be converted to 9-cisRA. Deletion of the putative PPAR/RXR site had no effect on induction of DDAH II in response to either atRA or 9-cisRA (Figure 5C). Deletion of promoter sequences upstream to this also had no effect on induction of DDAH II by atRA. In contrast, the deletion of promoter sequences downstream to this site abolished basal transcription of DDAH II, making it impossible to exclude an atRA response element within this region. Inhibition of protein synthesis using cycloheximide abolished the atRA-induced increase in DDAH II expression (Figure 5D).
Retinoic acid is important in the embryonic development of the cardiovascular system21 and can influence angiogenesis.1 It has recently been shown that atRA treatment may be beneficial in models of cardiovascular disease2–8,22,23⇓⇓⇓⇓⇓⇓⇓⇓ although the mechanisms involved are not understood. The present study demonstrates that atRA increases nitrite production by endothelial cells in a time-dependent manner and suggests that the upregulation of the enzyme DDAH II in endothelial cells contributes to this effect. These findings describe a novel vascular effect of atRA and clearly identify this retinoid as the first known transcriptional modulator of DDAH II.
A maximum increase in nitrite production by sEnd.1 cells (39.7±2.2%) was seen 24 hours after stimulation with atRA (n=9; P<0.001). atRA produces many of its effects by regulation of transcription and the time course over which atRA increased nitrite production was consistent with a genomic effect. We therefore studied the effects of atRA on NOS transcription. Neither nNOS nor iNOS mRNA was detectable in sEnd.1 cells and eNOS mRNA expression did not increase after atRA treatment. Indeed, a decrease in NOS expression after atRA treatment has previously been described in other cell types.24,25⇓
An alternative mechanism for the action of atRA might be the induction of another enzyme that indirectly influences the activity of expressed NOS. Our studies focused on those enzymes that regulate ADMA metabolism, namely the DDAH enzymes. ADMA is an endogenous and competitive inhibitor of NOS that is derived from the degradation of intracellular proteins. We and others have suggested that the metabolism of ADMA to citrulline by DDAH may play an important role in the regulation of NO synthesis.18,19⇓
DDAH I was expressed at low levels in endothelial cells and was not altered by atRA. However, we have recently identified a second isoform of DDAH (DDAH II) that is highly expressed in cardiovascular tissues and has a tissue distribution with similarities to that of eNOS.26 DDAH II expression was induced by atRA with the maximum increase seen 12 hours after atRA stimulation. A similar effect was seen in primary PAECs and the human cell lines SGHEC-7 and ECV304. Studies with actinomycin D confirmed that the effect was due to transcriptional regulation rather than the alteration of mRNA stability. This was consistent with the observation that in ECV304 cells transfected with a reporter construct driven by the human DDAH II promoter, atRA treatment increased promoter activity by 46.6±7.4%. Analysis of the promoter region of the DDAH II gene indicated the presence of a PPAR/RXR binding site, which might be a target for the action of atRA. Deletions of the PPAR/RXR site and sequences upstream to it had no effect on the induction of DDAH II by atRA. However, the deletion of sequences downstream to the PPAR/RXR site abolished basal levels of DDAH II transcription, making it impossible to exclude an atRA effect on this region. That atRA may be acting via an intermediate protein was suggested by the observation that cycloheximide treatment blocked the induction of DDAH II gene expression. These findings are consistent with the observation that a mouse cDNA sequence (7u), which we identified as DDAH II, is indirectly upregulated in growth-arrested melanoma cells treated with atRA.27 Establishing the precise molecular mechanism of this indirect atRA effect would be of interest because manipulation of ADMA levels is seen as a potential therapeutic approach in certain disease states.28
ADMA is a substrate for DDAH, whereas SDMA, also produced during protein turnover, is not. The ADMA/SDMA ratio therefore reflects DDAH activity and was decreased in cell culture medium after atRA treatment, consistent with an increase in DDAH activity. In order to establish whether the increased NO synthesis might be related to the induction of DDAH II and metabolism of ADMA, we used the DDAH inhibitor 4124W. In the present study, 4124W did not significantly affect nitrite production by unstimulated sEnd.1 cells, indicating that basal NO synthesis may not be regulated by DDAH activity in these cells. In contrast, 4124W significantly decreased nitrite production in atRA stimulated cells, reducing the increase in nitrite production attributable to atRA stimulation by 53.5±9.2%. At the concentration used, 4124W has previously been shown to inhibit purified DDAH activity by about 40% to 50%,18 and it is possible that complete inhibition of DDAH activity would reduce NO synthesis to baseline levels in stimulated endothelial cells. Alternatively, it is possible that other mechanisms may also contribute to the induction of NO synthesis by atRA. We cannot determine whether the induction of DDAH facilitates NO generation by ensuring that ADMA levels do not rise in response to increased protein turnover, or whether it causes a local fall in ADMA levels at the sites of NO generation. Either way, the experiments using the DDAH inhibitor show that induction of DDAH is essential for atRA-induced NO generation. Further studies will be required to determine whether DDAH induction drives or simply facilitates increased NO production.
The regulation of cardiovascular development and function by atRA is of potential physiological and therapeutic importance. Endothelial cells are exposed in vivo to relatively high concentrations (approximately 10−6 mol/L) of circulating retinol.29 The actions of atRA within the cell are regulated by cytoplasmic RA binding proteins and RA receptors, all of which are expressed and subject to transcriptional regulation in human endothelial cells.30 Wang et al12 have demonstrated that retinol can influence vascular smooth muscle function through an endothelium-dependent mechanism. The results of this study demonstrate that (1) atRA increases NO synthesis by endothelial cells despite no increase in eNOS mRNA expression, (2) atRA induces the expression of DDAH II by endothelial cells and reduces the ADMA/SDMA ratio, and (3) the inhibition of DDAH significantly reduces NO synthesis in atRA stimulated cells only. We infer from this that the regulation of DDAH II expression by atRA may in turn regulate NO synthesis by endothelial cells. Raised ADMA levels are associated with endothelial dysfunction and atherosclerosis,16,17⇓ and intracellular concentrations of ADMA are increased within endothelial cells that repopulate denuded areas after experimental balloon injury.31 Induction of DDAH II may lower ADMA levels and restore NO production in these and other conditions.
This work was supported by British Heart Foundation Program Grants RG94008 and RG2000007. The authors thank Dr J. Kabir for preparation of the PAECs.
↵*Both authors contributed equally to this work.
Original received September 5, 2001; resubmission received January 30, 2002; accepted February 21, 2002.
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- ↵Jang JJ, Kwan HH, Ho H-KV, Cooke JP, Fajardo LF. Asymmetric dimethylarginine: an endogenous anti-angiogenic factor. Circulation. 1998; 98 (suppl I): I-795.Abstract.
- ↵Boger RH, Bode-Boger SM, Szuba A, Tsao PS, Chan JR, Tangphao O, Blaschke TF, Cooke JP. Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Circulation. 1998; 98: 1842–1847.
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- ↵Colbert MC, Hall DG, Kimball TR, Witt SA, Lorenz JN, Kirby ML, Hewett TE, Klevitsky R, Robbins J. Cardiac compartment-specific overexpression of a modified retinoic acid receptor produces dilated cardiomyopathy and congestive heart failure in transgenic mice. J Clin Invest. 1997; 100: 1958–1968.
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- ↵Leiper J, Vallance P. Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res. 1999; 43: 542–548.
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