Published online before print April 19, 2007, doi: 10.1161/01.RES.0000267745.03488.47
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© 2007 American Heart Association, Inc.
Molecular Medicine |
C-Reactive Protein Downregulates Endothelial NO Synthase and Attenuates Reendothelialization In Vivo in Mice
From the Departments of Pediatrics (R.S., S.O.-L., L.H., L.L.G., A.K.G., W.Z., D.S., C.M., P.W.S.), Internal Medicine (W.V.), and Obstetrics and Gynecology (R.A.W.), University of Texas Southwestern Medical Center, Dallas; and Department of Biochemistry (S.B., D.S.), Case Western Reserve University School of Medicine, Cleveland, Ohio.
Correspondence to Philip W. Shaul, Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390. E-mail philip.shaul{at}utsouthwestern.edu
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Abstract |
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C-reactive protein (CRP) is an acute-phase reactant that is positively associated with cardiovascular disease risk and endothelial dysfunction. In cell culture, CRP decreases the expression of endothelial NO synthase (eNOS), which regulates diverse endothelial cell (EC) functions including migration. To determine whether CRP alters EC gene expression and phenotype in vivo, we studied CF1 transgenic mice expressing rabbit CRP (CF1-CRP) regulated by the phosphoenolpyruvate carboxykinase promoter such that levels could be altered by changing carbohydrate intake. Compared with CF1 controls with CRP of <1 µg/mL, carotid artery reendothelialization after perivascular electric injury was blunted in CF1-CRP mice, with CRP levels as low as 9 µg/mL. eNOS mRNA and enzyme abundance in carotid arteries was also blunted by CRP at 9 µg/mL in vivo, and ex vivo studies of isolated arteries showed that this occurs via direct action on the endothelium. The impaired reendothelialization with CRP was mimicked by NOS antagonism in CF1 mice; conversely, in cultured ECs CRP attenuation of migration was prevented by exogenous NO. Studies of EC transfected with human eNOS 5' flanking sequence fused to luciferase indicated that CRP decreases eNOS gene transcription. Both mutagenesis and electrophoretic mobility shift assays further revealed that CRP-responsive elements reside within the first 79 bp of the eNOS promoter. Thus, CRP downregulates eNOS and attenuates reendothelialization in vivo in mice, and this action of CRP on eNOS is mediated at the level of gene transcription.
Key Words: C-reactive protein • endothelial NO synthase • reendothelialization
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Introduction |
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C-reactive protein (CRP) is an acute-phase reactant and a member of the pentraxin family of proteins that classically serves as an opsonin and activates complement. Its hepatic synthesis is stimulated by interleukin-6 to yield levels that can rise 500-fold within 24 to 48 hours of the initiation of an inflammatory process.1–4 In addition to participating in immune response, CRP has received considerable attention as a risk factor for cardiovascular disease, with chronic modest elevations being associated with a greater likelihood of myocardial infarction, sudden cardiac death, stroke, and peripheral vascular disease.5–9 CRP is also a risk factor for the progression of subclinical vascular disease and for hypertension.10,11 Whether CRP plays a pathogenetic role in atherosclerosis is presently controversial.12,13
In vitro studies have shown that CRP decreases the expression of endothelial NO synthase (eNOS).14,15 In addition to a variety of other actions important to cardiovascular health, endothelium-derived NO promotes endothelial cell (EC) growth and migration and angiogenesis, which underlie both neovascularization and the maintenance of intimal layer integrity.16 An intact endothelial monolayer modulates hemostasis and thrombolysis and provides a nonpermeable barrier protecting vascular smooth muscle cells from circulating growth factors. Disruption of the intimal layer, either by gross denudation related to a vascular intervention or gap formation between ECs caused by disturbed shear stress, places the arterial wall at greater risk for vascular disease.17 Furthermore, whereas repeated EC removal worsens vascular lesion severity, enhanced reendothelialization attenuates lesion formation.18,19 CRP-induced attenuation of EC migration has been demonstrated in cell culture,15 suggesting that actions of the pentraxin on eNOS expression may result in changes in an EC phenotype of importance to vascular health. However, it is yet to be determined whether CRP modifies EC gene expression and phenotype in vivo.
The present study was designed to investigate the in vivo actions of CRP on endothelium in CRP transgenic mice. Mice expressing rabbit CRP under the regulation of the phosphoenolpyruvate carboxykinase (PEPCK) promoter on a CF1 background (CF1-CRP) were used to afford predictable, direct manipulation of CRP levels in vivo via changes in carbohydrate (CHO) intake.20 The use of CF1-CRP mice also avoids possible effects of contaminants or vehicle that may complicate studies with administered recombinant CRP.21–23 Using a perivascular electric injury model,24 we tested the hypothesis that CRP attenuates reendothelialization of the carotid artery in vivo. Additional studies were performed to answer the following questions. (1) Is reendothelialization impaired at clinically-relevant levels of CRP? (2) Is the effect on reendothelialization related to eNOS downregulation? (3) Do the effects of CRP on carotid artery eNOS entail systemic mechanisms or direct action on the endothelium? (4) How does CRP downregulate eNOS expression?
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Materials and Methods |
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Animal Model
Experiments were performed in CF1 and CF1-CRP mice that express a transgene consisting of the protein-coding region of the rabbit CRP gene linked to the promoter/regulatory region of the cytosolic form of the rat PEPCK gene.20 Mice hemizygous for the transgene (CF1-CRP–low) were also studied. Additional details are in the online data supplement at http://circres.ahajournals.org.
Carotid Artery Reendothelialization
Carotid artery reendothelialization was studied following perivascular electric injury.24 Additional details are in the online data supplement.
Quantification of eNOS mRNA
Real-time RT-PCR was performed to quantify eNOS mRNA abundance in noninjured carotid arteries of CF1 and CF1-CRP–low mice placed on the high CHO diet for 5 days.20 Additional details are in the online data supplement.
Quantification of NOS Activity
NOS enzymatic activity was determined in homogenates of noninjured carotid arteries from CF1 and CF1-CRP–low mice that received the high CHO diet for 5 days by measuring 14 C-L-arginine to 14 C-L-citrulline conversion.25 Further details are in the online data supplement. In additional experiments, carotid arteries from CF1 mice were incubated ex vivo in serum-free DMEM with human recombinant CRP (10 µg/mL, Calbiochem) for 18 hours. Control treatment consisted of CRP vehicle (140 mmol/L NaCl, 20 mmol/L Tris-HCl, 2 mmol/L CaCl2, 0.05%NaN3, pH 7.5) added at the same volume.26 A comparison of the characteristics of rabbit and human CRP is provided in the online data supplement.
Cell Culture
Primary bovine aortic ECs (BAECs) for use in EC migration assays were studied at passages 5 to 9.26 Primary ovine ECs for use in eNOS promoter activity assays were studied at passages 4 to 6.27
Endothelial Cell Migration Assay
The impact of CRP on the migration of BAECs was evaluated.24 Additional details are in the online data supplement. Heat-inactivated CRP26 did not alter migration (data not shown). In selected studies, cells were additionally treated with the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) (20 µmol/L).
eNOS Promoter Activity Assay
The impact of CRP on eNOS promoter activity was evaluated in ovine ECs using a promoter-reporter plasmid containing DNA from 1.6 kbp upstream of the eNOS AUG to the transcription start site inserted into the luciferase reporter gene plasmid pGL2 (–1624eNOS-Luc).27 Progressive 5' deletion mutants were also studied, which were designated –994eNOS-Luc, –318eNOS-Luc, –279eNOS-Luc, –248eNOS-Luc, and –79eNOS-Luc. Details are in the online data supplement.
Electrophoretic Mobility Shift Assays
Following treatment of ovine ECs with vehicle or CRP, nuclear extracts were prepared and incubated with a 32P-labeled oligonucleotide probe consisting of the 79 bp immediately upstream of the eNOS AUG.27 In competition studies, nonlabeled oligonucleotide probe was added at 200-fold molar excess before the addition of the 32P-labeled probe. Nuclear protein–DNA complexes were resolved on 5% nondenaturing polyacrylamide gels containing 1x Tris borate/EDTA buffer. Dried gels were exposed to Kodak XAR film for autoradiography.
Statistical Analysis
Data are presented as mean±SEM. Results between 2 groups were compared by Student’s t test. ANOVA with Neuman–Keuls post hoc testing was used to assess differences between 3 or more groups. Comparisons of the degree of reendothelialization between groups were done by Mann–Whitney tests. Significance was set at P<0.05.
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Results |
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Effect of CRP on Carotid Artery Reendothelialization
To first determine whether CRP affects reendothelialization, we performed perivascular electric injury experiments in 12- to 16-week-old male CF1 control versus CF1-CRP mice. Immediately following the injury procedure, the diet was switched from standard chow to a CHO-free, protein-rich chow to maximally activate transgene expression, and reendothelialization was assessed 5 days later. At the end of this regimen, CRP levels were <1 µg/mL in CF1 controls and CF1-CRP mice had CRP levels of 160±10 µg/mL (n=6/group). Compared with CF1 controls, reendothelialization was impaired in CF1-CRP mice (Figure 1A and 1B, respectively), as indicated by the larger area of remaining denudation that incorporated Evan’s blue dye in the latter group. Cumulative studies revealed that the area of remaining denudation was 83% greater in CF1-CRP versus CF1 (Figure 1C).
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To then determine whether CRP at lower levels also affects reendothelialization, additional experiments were performed in CF1 versus hemizygous CF1-CRP–low mice on high-CHO-containing diets to minimize transgene expression. This regimen yielded CRP levels of <1 µg/mL in CF1 mice and 9±5 µg/mL in CF1-CRP–low mice (n=6/group). Compared with CF1, reendothelialization was blunted in CF1-CRP–low mice (Figure 1D and E, respectively), and cumulative experiments indicated that the area of remaining denudation was 60% larger in CF1-CRP–low mice versus CF1 controls (Figure 1F).
Effect of CRP on eNOS Expression
It has been previously demonstrated in cultured ECs that CRP downregulates eNOS expression,14,15 and eNOS is critically involved in the regulation of EC migration.16 We therefore determined whether the impaired reendothelialization is associated with a change in eNOS expression. First, steady-state eNOS mRNA levels were quantified in noninjured carotid arteries from CF1 versus CF1-CRP–low mice following 5 days placement on the high-CHO-containing chow that yields CRP levels of <1 µg/mL and 9 µg/mL, respectively. Using real-time RT-PCR, it was found that eNOS mRNA abundance was decreased by 74% by CRP (Figure 2A). In parallel, NOS enzymatic activity was decreased by 53% in the carotid arteries of CF1-CRP–low mice versus CF1 controls (Figure 2B). Thus, CRP causes a decline in both eNOS mRNA and enzyme abundance in vivo in mice.
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P<0.05 vs eNOS.To determine whether the reduction in eNOS in CF1-CRP–low mice is attributable to systemic actions of CRP or a direct effect of the pentraxin on the vasculature, ex vivo experiments were performed with carotid arteries from CF1 mice. Arteries were incubated under control conditions or in the presence of CRP for 18 hours, and NOS enzymatic activity was evaluated in a manner that distinguished eNOS- and inducible NOS (iNOS)-derived activity based on calcium dependence. CRP had no effect on iNOS-derived NOS activity, which was less than eNOS-derived activity in control-treated arteries. However, paralleling the decline in eNOS caused by CRP in vivo, artery incubation with CRP caused a 90% decrease in eNOS-derived enzymatic activity (Figure 2C). In contrast, heat-inactivated CRP26 caused no change in eNOS-derived or iNOS-derived enzyme activity (91±11% of control activity and 101±13% of control activity, respectively). Thus, CRP directly reduces arterial eNOS expression, and the changes in eNOS observed in vivo are not caused by CRP-induced systemic actions.
CRP, NO Deficiency, and Impaired Endothelial Cell Migration
Having demonstrated that CRP causes eNOS downregulation in association with impaired reendothelialization in vivo, we next determined whether the blunted reendothelialization phenotype observed in CF1-CRP mice is mimicked in the same mouse strain by NO deficiency. CF1 mice were administered drinking water or drinking water plus NG-nitro-L-arginine methyl ester (L-NAME), perivascular electric injury was performed, and the area of remaining denudation was evaluated 5 days later. Compared with control mice, reendothelialization was impaired in L-NAME–treated mice (Figure 3A and 3B, respectively). Cumulative studies revealed that the area of remaining denudation was 170% larger in L-NAME–treated versus control-treated mice (Figure 3C).
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To provide additional linkage among CRP, NO deficiency, and blunted EC migration, migration assays were performed with cultured BAECs. Compared with control-treated cells, migration was stimulated by vascular endothelial growth factor (VEGF) treatment (Figure 4A and 4B, respectively), and VEGF-stimulated migration was attenuated by CRP (Figure 4C). However, cotreatment with the NO donor SNAP rescued normal VEGF-stimulated migration in the presence of CRP (Figure 4D). Findings were confirmed in repeat studies, and the cumulative data are shown in Figure 4E. eNOS downregulation by CRP was also demonstrable (Figure 4F). The collective observations made in vivo and in cell culture indicate that NO deficiency underlies the negative impact of CRP on EC migration.
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Effect of CRP on eNOS Promoter Activity
To further understand the molecular basis of eNOS downregulation by CRP, we determined whether the pentraxin modified eNOS transcription in studies of the activity of the human eNOS promoter transfected into ovine ECs. Using the full-length promoter (–1624eNOS-Luc), we found that treatment with CRP for 6 hours causes a decline in basal promoter activity (Figure 5A). More substantial declines in promoter activity were observed with 18 hours of CRP treatment. Polymyxin B did not alter the effect of CRP on the eNOS promoter.
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The dose response to CRP was also evaluated (Figure 5B). CRP concentrations of 10 µg/mL or more caused declines in eNOS promoter activity. In studies performed at 50 µg/mL, recombinant and ascites-derived human CRP caused comparable 40±3% and 46±2% declines in eNOS promoter activity, respectively. Declines in eNOS protein expression were also apparent, particularly with CRP treatment at 10 µg/mL or more (Figure 5B, inset). Thus, paralleling the in vivo findings for CRP-induced downregulation of eNOS expression (Figure 2), eNOS gene transcription was blunted by CRP.
Promoter Elements Required for eNOS Regulation by CRP
To determine the elements in the eNOS promoter required for CRP responsiveness, the activities of progressive 5' deletion mutants were evaluated. Basal promoter activity in the absence of CRP did not change with deletion from –1624 to –994, nor with further deletion to –318. In contrast, basal promoter activity rose 5.6±1.6-fold (P<0.05) with deletion to –279 compared with full-length promoter, which is consistent with our prior findings regarding this region of the promoter in studies of eNOS regulation in airway epithelium.27 In experiments comparing the CRP responsiveness of –1624eNOS-Luc, –994eNOS-Luc, –318eNOS-Luc, and –279eNOS-Luc there were equivalent 50% declines in activity in the presence of the pentraxin (Figure 6A). These findings indicate that the promoter elements responsible for CRP action reside more proximal in the eNOS 5' flanking sequence than –279.
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To evaluate this further, additional 5' deletion mutants were studied. With deletion from –1624 to –248, basal promoter activity did not change, nor did it change with further deletion to –79. In comparisons of the impact of CRP on the activities of –1624eNOS-Luc, –248eNOS-Luc, and –79eNOS-Luc, there were equivalent 40% to 50% declines in activity in the presence of the pentraxin (Figure 6B). Thus, the CRP-responsive regulatory elements are located within 79 bp of the eNOS transcription initiation site.
The region of the eNOS promoter residing between –79 and –1 does not contain regulatory elements known to participate in eNOS gene regulation.28,29 We therefore performed electrophoretic mobility shift assays to provide an independent means to determine whether sequences within –79 to –1 are of relevance to CRP modulation of eNOS. Nuclear extracts from vehicle versus CRP-treated (10 µg/mL for 24 hours) ovine ECs were incubated with a double-stranded oligonucleotide probe consisting of the 79 bp immediately upstream of the eNOS AUG. Nuclei from control cells displayed one major DNA–protein complex (Figure 7A, arrowhead). In contrast, nuclei from CRP-treated cells yielded 2 slower-migrating complexes (Figure 7A, arrows). The complexes were diminished by a 200-fold molar excess of unlabeled probe (Figure 7B). Thus, CRP induces nuclear protein binding to the same proximal region of the eNOS promoter that displays CRP responsiveness in promoter-reporter assays.
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Discussion |
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Studies of cultured ECs indicate that CRP modifies the expression of important EC genes and also alters diverse EC functions.13 This has particularly been elucidated regarding the downregulation of eNOS expression and EC migration.13–15 In the present study, we demonstrated in CRP transgenic mice that the pentraxin causes impaired reendothelialization and blunted eNOS expression in the carotid artery, indicating that both endothelial gene expression and a critical ECs phenotype are altered by CRP in vivo.
We first demonstrated that maximal transgene activation in CF1-CRP mice induced by a CHO-free, high-protein diet, which yields CRP levels of 160 µg/mL, causes impaired reendothelialization. Minimal transgene activation was then accomplished in hemizygous CF1-CRP–low mice on a CHO-rich diet, resulting in CRP levels of 9 µg/mL, and blunted reendothelialization was still observed. This finding indicates that modest elevations in CRP that are associated with greater cardiovascular disease risk in otherwise healthy individuals in the absence of acute inflammation or infection30 have adverse effects on an important EC phenotype in mice.
To determine whether the in vivo effect of CRP on reendothelialization of the carotid artery is related to a decline in eNOS expression, eNOS mRNA and NOS enzymatic activity were evaluated in the carotid arteries of hemizygous CF1-CRP–low mice with CRP levels of 9 µg/mL. Both transcript abundance and enzyme activity were attenuated by CRP, consistent with previous studies in which CRP caused declines in eNOS mRNA and protein expression in cultured ECs.14,15 Mirroring the in vivo findings with the rabbit CRP transgene, ex vivo incubations of mouse carotid arteries with recombinant human CRP performed in the absence of serum also yielded a decline in eNOS abundance. This is not surprising because the molecular structure, binding characteristics, and functions of CRP are similar for the rabbit and human proteins (see the online data supplement). Using multiple strategies including heat inactivation of the recombinant protein, it was further revealed that the ex vivo observation was attributable to the actions of CRP and not contaminants or the vehicles for the CRP preparations. Although it has been a point of debate, such strategies have recently been used to exclude the involvement of contaminants or vehicle in multiple changes in gene expression and function induced by recombinant CRP in cultured ECs.13 From a mechanistic standpoint, the parallel in vivo and ex vivo observations further indicate that classical actions of CRP entailing the activation of complement or signaling in monocytes or other immune response cells1,2 do not play a role in CRP downregulation of eNOS. These cumulative results instead indicate that CRP has a direct effect on gene expression in endothelium in its native state.
To determine whether the loss in NOS activity in the carotid arteries of CF1-CRP–low mice versus CF1 controls may underlie the blunted reendothelialization observed with CRP in vivo, L-NAME was administered to CF1 mice and reendothelialization was evaluated. The phenotype of impaired reendothelialization seen with transgenic CRP elevation was replicated by NOS antagonism in the same mouse strain. Furthermore, in the EC migration model using BAECs in culture, blunted migration with CRP was rescued by the provision of exogenous NO. These combined observations suggest that the basis for CRP antagonism of reendothelialization lies in the downregulation of eNOS expression and the resulting decrease in bioavailable NO that is critical to the promotion of EC growth and migration.16 When considered along with our prior work demonstrating that CRP also antagonizes eNOS activation by diverse agonists,26 it is apparent that the mechanisms by which CRP leads to a loss of NO are multiple. Further studies are now indicated to determine whether CRP impairs the function of endothelial progenitor cells in vivo, which are released from the bone marrow in an NO-dependent manner to participate in various forms of neovascularization31,32 and which are negatively influenced by CRP in cell culture studies.33
In light of the well-recognized direct roles of endothelium-derived NO in the regulation of blood pressure, in the promotion of vasodilation and in the attenuation of thrombosis,34 the current in vivo observation of eNOS downregulation by CRP may be relevant to the greater risk of hypertension and the blunted vascular responses to acetylcholine seen with elevated levels of CRP in humans,10,35 and also the exaggerated thrombosis reported in CRP transgenic mice.36 We recently reported that the CRP transgenic mice have systolic hypertension and supersensitivity to angiotensin II caused by a loss of vascular angiotensin receptor subtype 2 expression that is likely attributable to NO deficiency.37 In contrast, despite considerable evidence for a role for inflammation in atherogenesis,38 care should be taken in the extrapolation of the present findings to that disorder. Findings regarding the impact of CRP on atherosclerosis in apolipoprotein E–null mice and in hyperlipidemic rabbits have been mixed.12 Future studies of the contribution of CRP to the pathogenesis of specific cardiovascular conditions in mice should be undertaken with the recognition that, although found to not be pertinent to the present investigation, there is uncertainty whether CRP activation of complement or signaling in immune response cells is fully operative in mice.12
Having demonstrated that CRP antagonism of eNOS expression occurs not only in cell culture but also in vivo, the molecular mechanisms underlying eNOS downregulation were pursued. In prior studies, CRP-induced declines in steady-state eNOS mRNA levels in cultured ECs were attributed to a 16% decrease in eNOS mRNA half-life.14 Considering that the impact of CRP on eNOS transcript abundance both in vitro14 and now in vivo exceeds the relative change reported in mRNA degradation, the effect of CRP on eNOS promoter activity was investigated. At CRP levels of 10 µg/mL and greater, the pentraxin caused a 40% to 50% decrease in promoter activity in parallel with a decline in eNOS protein abundance, indicating that a considerable portion of the eNOS downregulation occurs at the level of gene transcription. Studies of deletion mutants of the eNOS promoter further revealed that the CRP-responsive regulatory element(s) is located within 79 bp of the 5' flanking sequence of the eNOS gene. The role of the proximal promoter in CRP action was then confirmed in electrophoretic mobility shift assays that demonstrated that the pentraxin induces nuclear protein binding to the same region. Because the region of the eNOS promoter residing between –79 and –1 does not contain sequences known to participate in eNOS gene regulation,28,29 studies to identify novel regulatory elements mediating CRP responsiveness are now indicated.
Pioneering experiments in cell culture have shown that multiple EC genes in addition to eNOS are likely to be modified by CRP. It has been found that CRP induces the expression of intracellular and vascular cell adhesion molecules, monocyte chemoattractant protein-1, E-selectin, the receptor for advanced glycation end products (RAGE), oxidized low-density lipoprotein receptor-1, CD40/CD40 ligand, plasminogen activator inhibitor-1, and tissue plasminogen activator, and it induces the release of endothelin-1 and interleukin-8. Furthermore, CRP impairs the expression of thrombomodulin and endothelial protein C receptor and the production of prostacyclin.13 Other studies performed in vitro and in vivo have demonstrated that CRP binds to and alters EC phenotype via Fc receptors, in particular CD32 (Fc receptor IIA/B) and CD64 (Fc receptor I), and that it upregulates CD32 and CD64 surface expression in cultured ECs,13,26 thereby potentially amplifying its own actions. Future studies of endothelial Fc receptors will provide greater understanding of the basis for CRP regulation of gene expression in endothelium in vivo.
Paralleling prior investigations of eNOS expression and action in cultured endothelium, the present studies in transgenic mice demonstrate that CRP alters EC gene expression and phenotype in vivo. These findings provide further evidence that CRP is a potential mediator of vascular dysfunction and not solely a marker of concomitant inflammation. Direct impact of CRP on EC gene transcription is also documented. Further studies in this realm will help to clarify the contribution of CRP to the pathogenesis of vascular disorders.
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Acknowledgments |
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Sources of Funding
This work was supported by NIH grants HL75473 (to P.W.S.) and AG02467 (to D.S.), by the Donald W. Reynold’s Cardiovascular Clinical Research Center (to W.V.), and by the Crystal Charity Ball Center for Pediatric Critical Care Research and the Lowe Foundation (to P.W.S.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received December 26, 2006; revision received March 20, 2007; accepted April 10, 2007.
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