FcRIIB Mediates C-Reactive Protein Inhibition of Endothelial NO Synthase
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循环研究杂志 2005年第11期
the Departments of Pediatrics (C.M., A.K.G., I.S.Y., S.O.-L., L.L.G., L.H., P.W.S.) and Internal Medicine (R.V.S., D.R.K., G.D.T.), University of Texas Southwestern Medical Center, Dallas
Department of Medicine (J.E.S.), Cornell University Weill Medical College, New York, NY
Department of Biochemistry (S.B., D.S.), Case Western Reserve University School of Medicine, Cleveland, Ohio.
Abstract
C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fc receptors, causes parallel okadaic acideCsensitive loss of eNOS function, FcRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcRIIB. In FcRIIB+/+ mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcRIIBeC/eC mice. Thus FcRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.
Key Words: C-reactive protein endothelial NO synthase Fc receptor PP2A
Introduction
C-reactive protein (CRP) is an acute-phase reactant and a member of the pentraxin family of proteins. 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. CRP serves as an opsonin and activates complement by binding to C1q.1eC4
In addition to participating in immune response, CRP has received considerable attention as a risk factor for cardiovascular disease. Although the relative predictive value of CRP versus other risk factors has been variable, the finding that CRP levels correlate with cardiovascular disease has been remarkably consistent across populations.5eC9 CRP is also a risk factor for the progression of subclinical vascular disease and for hypertension.10,11 Furthermore, a primary effect of CRP on endothelium is plausible because elevated levels are associated with endothelial dysfunction, as evidenced by blunted forearm vascular responses to acetylcholine (Ach), which activates endothelial NO synthase (eNOS) to generate NO on L-arginine conversion to L-citrulline.12 Potentially consistent with these clinical observations, CRP transgenic mice have exaggerated thrombosis,13 and CRP blunts eNOS expression and function in cultured endothelial cells.14,15 However, it has yet to be determined whether CRP has direct effects on vascular endothelium in vivo, and the basis for such effects is unknown.
In the present study, we investigated the mechanisms underlying CRP actions on endothelium by testing the hypothesis that CRP attenuates eNOS activation in cultured endothelial cells. The resulting effect on monocyte adhesion was also determined. Because eNOS activation entails phosphatidylinositol 3-kinaseeCmediated increases in Ser1179 phosphorylation, which are counter-regulated by the phosphatase PP2A,16eC18 alterations in Ser1179 phosphorylation and the potential involvement of PP2A were investigated. To further identify the basis for this process, additional experiments tested the role of Fc receptors for IgG, which display high affinity for CRP and modulate CRP actions in immune response cells.19eC23 Moreover, studies of CRP-induced changes in endothelial function were performed in mice to delineate whether these mechanisms are operative in vivo.
Materials and Methods
Cell Culture and Transfection
Primary bovine aortic endothelial cells (BAEC) and human aortic endothelial cells (HAEC) (Cambrex Corp) were used within 7 passages. MFLM-4 were provided by Dr Ann Akeson (Children’s Hospital Medical Center, Cincinnati, Ohio). To study the role of FcRIIB, FcRII-negative COS-7 cells were transfected with human FcRIIB1 cDNA (a gift from Dr Catherine Sautes-Fridman, Paris, France),24 selected with Zeocin (Invitrogen), and cloned by limiting dilution. FcRIIB expression was tested by fluorescence-activated cell sorting (FACS) with the monoclonal antibody AT10 (provided by Dr P.M. Morganelli, White River Junction, Vermont). Zeocin-resistant cells not expressing detectable Fc receptor (FcR) served as controls.
eNOS Activation Assays
eNOS activation was assessed in whole cells by measuring [3H]L-arginine conversion to [3H]L-citrulline.25 Cell treatments included human recombinant CRP (Calbiochem), ascites-derived human CRP, purified and characterized as previously described,26 or human recombinant serum amyloid P component (SAP) (Calbiochem) added during a 15-minute preincubation and the 15-minute incubation for eNOS activation. For additional details, see the online data supplement available at http://circres.ahajournals.org. Control cells were exposed to CRP or SAP heated at 100°C for 60 minutes. Stimulated activity is expressed as percentage of basal activity, and results were confirmed in 3 experiments. eNOS activation was also evaluated ex vivo in isolated carotid arteries from 10- to 12-week-old male C57BL/6 mice by measuring cGMP accumulation during 2-minute incubations.27 The care and use of all study animals was approved by the International Animal Care and Use Committee at the University of Texas Southwestern Medical Center.
Monocyte Adhesion Assays
The adhesion of U937 cells to monolayers of BAEC was evaluated as previously described.28 Following U937 and endothelial cell coincubation and washing, cells were fixed and the number of adherent cells was counted. See the online data supplement for additional details.
Short-Interference RNA Preparation and Transfection
Double-stranded RNA with sequence 5'-CCAAGCUGCAAUCAUGGAA-3' was designed to target the open reading frame of the bovine PP2A catalytic subunit C29 (GenBank accession no. M16968). Scrambled sequence served as control. BAEC were transfected with 80 nmol/L RNA as described previously,30 and PP2A expression and eNOS activation were assessed 48 hours posttransfection.
Immunoblot Analyses
Immunoblots were performed to assess eNOS phosphorylation using antieCphospho-Ser1179 eNOS antibody (Cell Signaling Technology) and total eNOS abundance using eNOS monoclonal antibody (BD Biosciences Pharmingen).31 BAEC were starved overnight in the absence of serum or phenol red in DMEM before eNOS agonist treatment. Results shown were confirmed in 3 independent experiments.
RT-PCR for FcRIIA/B in Endothelium
FcRIIA/B expression was evaluated in HAEC by RT-PCR using Raji cells as positive controls. To assess and quantitate receptor expression in native endothelium, real-time RT-PCR studies were performed using RNA from endothelial cells isolated from the aorta, carotid artery, and heart of Tie2eCgreen fluorescent protein (GFP) transgenic mice by FACS analysis.32 See the online data supplement for additional details.
Carotid Artery Vascular Conductance in Mice
To determine whether CRP alters signaling to eNOS in vivo, Ach-induced changes in carotid artery vascular conductance were measured shortly before and after IP vehicle or CRP administration in mice.33 Studies were performed at 10 to 12 weeks of age in male C57BL/6 mice or in male FcRIIB+/+ versus FcRIIBeC/eC B6:129S mice (Jackson Laboratory).34 The dose of CRP used (250 e IP) yielded serum levels of 38±4 e/mL (n=6). See the online data supplement for additional details.
Statistical Analysis
Student t tests or ANOVA with NeumaneCKeuls post hoc testing were used to assess differences between 2 groups or among more than 2 groups, respectively, and significance was set at P<0.05.
Results
CRP Antagonism of eNOS Activation
To determine whether CRP alters eNOS activation, BAEC or HAEC were preincubated with heat-treated (control) or nontreated recombinant human CRP (5 e/mL), and eNOS stimulation was evaluated (Figure 1, left and right for BAEC and HAEC, respectively). Vascular endothelial growth factor (VEGF) stimulation of eNOS in either BAEC or HAEC was attenuated by CRP (Figure 1A). Similarly, CRP blunted eNOS activation by high-density lipoprotein (HDL) (Figure 1B) and by insulin (Figure 1C). Thus, CRP attenuates eNOS activation mediated by diverse agonists, intact CRP protein is required, and the process occurs in bovine and human endothelium.
Lipopolysaccharide (LPS) was not responsible for the observed effects on eNOS. Whereas CRP (5 e/mL) potently attenuated VEGF-stimulated eNOS activity, LPS (5 e/mL) had no effect (Figure 2A). In addition, human recombinant CRP and ascites-derived CRP26 caused comparable inhibition of eNOS activation (Figure 2B). SAP, the related pentraxin that is 60% homologous to CRP,35 also blocked eNOS activation (Figure 2C), and dose-response studies showed CRP effects at 5 e/mL and SAP effects at 2 e/mL (Figure 2D). These findings indicate that levels of CRP that have been associated with the risk of cardiovascular disease (3 to 10 e/mL)1 are operative and that the capacity to block eNOS activation is shared by CRP and SAP.
To determine whether CRP actions on eNOS alter endothelial cell function, the impact on monocyte adhesion was evaluated. LPS caused a marked increase in monocyte adhesion compared with control conditions (Figure 3A and 3B, respectively); this was reversed by the eNOS agonist insulin (Figure 3C), and the effect of insulin was confirmed to be NO dependent using NG-nitro-L-arginine methyl ester (L-NAME) (Figure 3D). CRP (3 e/mL) prevented the lessening of adhesion with insulin (Figure 3E), and this was not related to a change in eNOS expression (data not shown). The impact of CRP on adhesion was fully reversed by S-nitroso-N-acetyl-D, L-penicillamine (Figure 3F). Thus, CRP-induced declines in NO production promote monocyte adhesion to endothelium.
eNOS Phosphorylation and Involvement of PP2A
To define the mechanisms underlying CRP antagonism of eNOS, we first determined whether changes in gene transcription are involved. CRP caused comparable blockade of VEGF activation of eNOS in control and actinomycin DeCtreated cells (Figure 4A), indicating that CRP action is transcription independent. Because eNOS stimulation entails phosphatidylinositol 3-kinaseeCmediated increases in Ser1179 phosphorylation,17 changes in phosphorylation of the enzyme were investigated. Using HDL as agonist, an increase in phosphorylated eNOS occurred in control cells but not in CRP-treated cells (Figure 4B, top). Under these conditions, CRP exposure did not alter total eNOS abundance. The increase in eNOS phosphorylation by insulin was similarly blunted by CRP (Figure 4B, bottom), and comparable findings were obtained with estradiol-17 (10 nmol/L) (data not shown) or VEGF as agonist (Figure 4C). Because PP2A controls eNOS phosphorylation at Ser1179,16,18 the effect of the selective PP2A inhibitor okadaic acid (100 nmol/L) was investigated. In contrast to the diminution in eNOS phosphorylation observed with CRP alone, okadaic acid rescued Ser1179 phosphorylation in the presence of CRP (Figure 4C). In parallel, VEGF-stimulated eNOS activity was rescued by okadaic acid (Figure 4D). To confirm involvement of PP2A, short-interference RNA (siRNA) was used to diminish expression of the phosphatase in BAEC. With a 53% decline in PP2A protein expression (Figure 4E, inset), there was a parallel 48% rescue of eNOS activation by VEGF in the presence of CRP (Figure 4E). We also determined whether CRP blunts eNOS stimulation by bradykinin, which entails intracellular calcium elevation and eNOS phosphorylation.36 CRP inhibited bradykinin-induced eNOS phosphorylation and activation in an okadaic acideCsensitive manner (Figure 4F and 4G, respectively). These cumulative findings indicate that the negative modulation of eNOS by CRP is mediated by PP2A-induced changes in phosphorylation.
CRP Action In Vivo
In preparation for in vivo studies of CRP action in mice, the effect of CRP on Ach-mediated eNOS activation was evaluated in MFLM-4 mouse endothelial cells. eNOS stimulation by Ach was absent in CRP-treated cells (Figure 5A). CRP also caused blunted cGMP accumulation with Ach in isolated mouse carotid arteries (Figure 5B). In contrast, cGMP accumulation with the NO donor sodium nitroprusside was not altered by CRP (data not shown). Thus, CRP antagonizes Ach activation of eNOS ex vivo in mouse endothelium. Paralleling the findings with other agonists, CRP inhibited Ach-induced eNOS phosphorylation and activation in an okadaic acideCsensitive manner (Figure 5C and 5D, respectively). Thus, CRP blockade of signaling by Ach typifies the impact of CRP on multiple mediators of endothelial function.
To determine whether CRP attenuates eNOS activation in vivo, Ach-induced changes in carotid artery vascular conductance were measured in mice.33 Whereas the control vehicle had no effect (Figure 5E, left), following CRP administration the Ach response was blunted by 50% (Figure 5E, right). Thus, CRP actions on eNOS observed in cultured endothelium also occur in endothelium in vivo.
Role of FcRs
If the actions of CRP on eNOS require FcRs, which display high affinity for CRP and mediate its effects in immune response cells,1,19eC23 FcR crosslinking should yield the same phenotype as CRP. Aggregated IgG (aIgG) caused a concentration-dependent diminution in eNOS activation by VEGF (Figure 6A). In addition, aIgG blunted Ser1179 phosphorylation and eNOS activation in response to VEGF in an okadaic acideCsensitive manner (Figure 6B and 6C). These findings indicate that FcRs modify eNOS function via the activation of PP2A.
We then determined whether FcRIIs, the principal high-affinity receptors for CRP, are expressed in endothelium. In humans, FcRIIA is an activation receptor, and FcRIIB is an inhibitory receptor, and only FcRIIB has been identified in mice.37 In studies of HAEC, RT-PCR demonstrated mRNA expression for FcRIIB but not FcRIIA (Figure 6D). To determine whether FcRIIB is expressed in endothelium in vivo, endothelial cells were purified by FACS analysis from the aorta, carotid artery, and heart of Tie2-GFP transgenic mice.32 Following RNA isolation and amplification, quantitative real-time RT-PCR showed that the abundance of mRNA for eNOS, the target of interest for CRP action, was greater in endothelium from aorta and carotid artery compared with cardiac endothelium (Figure 6E, top). FcRIIB mRNA was detected in greater abundance in aortic and cardiac endothelium compared with carotid artery endothelium (Figure 6E, bottom). Thus, FcRIIB is expressed in human endothelial cells and in mouse endothelium, and the level of expression varies between blood vessel types.
The causal role of FcRIIB in CRP antagonism of eNOS was then tested in COS-7 cells expressing eNOS and scavenger receptor B type I (SR-BI) to enable eNOS activation by HDL. In control cells not expressing FcRIIB, CRP did not antagonize eNOS activation (Figure 7A, left). In contrast, in cells expressing FcRIIB, CRP blunted eNOS activation (Figure 7A, right), indicating that the action of CRP requires FcRIIB. CRP binding specifically to FcRIIB in this system was confirmed by FACS analysis (data not shown). The requirement for FcRIIB was also tested in vivo in comparisons of Ach-mediated increases in carotid vascular conductance in FcRIIB+/+ and FcRIIBeC/eC mice. Control vehicle did not alter Ach responses in either FcRIIB+/+ or FcRIIBeC/eC mice (Figure 7B), and Ach-induced increases in conductance were attenuated by CRP in FcRIIB+/+ mice (Figure 7C, left). In sharp contrast, CRP did not blunt Ach responses in FcRIIBeC/eC, and instead Ach-induced increases in conductance were enhanced by CRP (Figure 7C, right). These in vivo findings confirm that FcRIIB is required for CRP inhibition of eNOS. Interestingly, because there was actual enhancement of the Ach vasodilatory response by CRP in FcRIIBeC/eC mice, other mechanisms of CRP action may have been unmasked in the absence of FcRIIB. We postulate that the latter processes may involve stimulatory FcRs such as FcRIII, which cause increases in intracellular calcium on their activation and thereby would potentially enhance eNOS activity.37
Discussion
CRP levels are strongly correlated with increased risk for cardiovascular disease and with endothelial dysfunction related to decreased NO bioavailability.5eC12 However, the basis for potential effects of CRP on the endothelium has been unclear. Here we show that CRP causes potent antagonism of eNOS activation by diverse agonists resulting from changes in eNOS phosphorylation mediated by PP2A. Importantly, eNOS antagonism occurs at levels of CRP that have been associated with the risk of cardiovascular disease.1 When these processes are considered along with the previously known action of CRP to attenuate eNOS expression following prolonged exposure (24 hours),14,15 it is apparent that CRP decreases NO bioavailability by multiple mechanisms.
To link the effect of CRP on eNOS activation to a change in endothelial cell function, we show that CRP-induced declines in NO production underlie the promotion of monocyte adhesion by CRP in vitro. We also demonstrate in a mouse model that CRP antagonism of eNOS is operative in vivo. Such findings provide an explanation for the more than 50% decline in endothelium-dependent vasodilation that was recently observed following CRP infusion in hypercholesterolemic patients (E.S.G. Stroes, personal communication, 2005). Because there are multiple lines of evidence indicating that a loss in endothelial NO production plays a critical role in the pathogenesis of cardiovascular disease,38 we further propose that the resulting diminution in NO production may underlie the increased long-term cardiovascular risk associated with higher CRP levels in the absence of acute inflammation,8,39 as well as the poorer prognosis associated with even greater elevations in CRP during acute events.40 Whereas enhanced thrombosis has been effectively demonstrated in CRP transgenic mice,13 it is less clear from mouse models whether CRP accelerates atherogenesis.41eC43 Considering the complexity and diversity of cardiovascular diseases and their etiologies, further studies of the direct impact of eNOS-related CRP actions on vascular health and disease in animal models are now warranted.
In addition to the observed actions of CRP on eNOS, we found that the related pentraxin SAP had comparable effect at physiologic levels. SAP is a major acute-phase reactant in the mouse and a constitutive protein in the blood of humans, with basal concentrations of 2 to 10 and 40 e/mL, respectively.44,45 To date, SAP has not been associated with defects in vascular function. Although saturable binding of SAP to the IgG receptor subclass Fc, in particular FcRI, FcRIIa, and FcRIIIb, has been described in transfected COS cells,3 SAP may not be accessible to the endothelium in vivo because of association with various proteins in the plasma or vasculature.46eC48 More studies will be required to elucidate the role of SAP in vascular biology.
The known actions of CRP in immune-response cells are mediated by Fc receptors, with FcRII acting as the principal high-affinity receptor.19eC23 We demonstrate that human endothelial cells in culture and mouse endothelial cells in their native context express mRNA for FcRIIB. Attempts to detect FcRIIB protein in endothelium have been hindered by the lack of specificity of available antibodies and the low level of receptor abundance under quiescent conditions.49 However, using both gain-of-function and loss-of-function strategies in vitro and in vivo, we demonstrate that FcRIIB underlies the actions of CRP on vascular endothelium. Our studies are also the first to mechanistically link FcRIIB to PP2A in any paradigm, and the basis for the coupling of FcRIIB with PP2A warrants further investigation. Just as importantly, we show that the classical ligand for Fc receptors, aIgG, has identical action to CRP in endothelium. It is well established that patients with rheumatoid arthritis and systemic lupus erythematosus have a higher incidence of cardiovascular disease and endothelial dysfunction that is not explained by traditional risk factors.50eC54 We propose that elevated levels of CRP or circulating immune complexes engage endothelial FcRIIB to attenuate eNOS activity in these patients, thereby adversely affecting endothelial function and possibly contributing to their greater cardiovascular disease risk.
Collectively the present observations reveal a novel series of mechanisms by which CRP is a direct mediator of endothelial dysfunction. Our findings provide a new framework for understanding how CRP, and also circulating immune complexes, may contribute to the pathogenesis of vascular disease. It is anticipated that further research in this realm will lead to new prophylactic and therapeutic strategies to combat the vascular complications of multiple inflammatory and autoimmune disorders
Acknowledgments
This work was supported by NIH grants HL75473 (to P.W.S.), HL06296 (to G.D.T.), and AG02467 (to D.S.). The project was also supported by American Heart Association Scientist Development Award 0235107N (to C.M.), the Children’s Medical Center Dallas Foundation (to A.K.G.), the Crystal Charity Ball Center for Pediatric Critical Care Research and the Lowe Foundation (to P.W.S.), and the Simmons Family Foundation (to D.R.K.). We are indebted to Miranda King for technical assistance and to Marilyn Dixon for assistance in preparing the manuscript.
Both authors contributed equally to this study.
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Department of Medicine (J.E.S.), Cornell University Weill Medical College, New York, NY
Department of Biochemistry (S.B., D.S.), Case Western Reserve University School of Medicine, Cleveland, Ohio.
Abstract
C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk and endothelial dysfunction. Whether CRP has direct actions on endothelium and the mechanisms underlying such actions are unknown. Here we show in cultured endothelium that CRP prevents endothelial NO synthase (eNOS) activation by diverse agonists, resulting in the promotion of monocyte adhesion. CRP antagonism of eNOS occurs nongenomically and is attributable to blunted eNOS phosphorylation at Ser1179. Okadaic acid or knockdown of PP2A by short-interference RNA reverses CRP antagonism of eNOS, indicating a key role for the phosphatase. Aggregated IgG, the known ligand for Fc receptors, causes parallel okadaic acideCsensitive loss of eNOS function, FcRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcRIIB. In FcRIIB+/+ mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcRIIBeC/eC mice. Thus FcRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.
Key Words: C-reactive protein endothelial NO synthase Fc receptor PP2A
Introduction
C-reactive protein (CRP) is an acute-phase reactant and a member of the pentraxin family of proteins. 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. CRP serves as an opsonin and activates complement by binding to C1q.1eC4
In addition to participating in immune response, CRP has received considerable attention as a risk factor for cardiovascular disease. Although the relative predictive value of CRP versus other risk factors has been variable, the finding that CRP levels correlate with cardiovascular disease has been remarkably consistent across populations.5eC9 CRP is also a risk factor for the progression of subclinical vascular disease and for hypertension.10,11 Furthermore, a primary effect of CRP on endothelium is plausible because elevated levels are associated with endothelial dysfunction, as evidenced by blunted forearm vascular responses to acetylcholine (Ach), which activates endothelial NO synthase (eNOS) to generate NO on L-arginine conversion to L-citrulline.12 Potentially consistent with these clinical observations, CRP transgenic mice have exaggerated thrombosis,13 and CRP blunts eNOS expression and function in cultured endothelial cells.14,15 However, it has yet to be determined whether CRP has direct effects on vascular endothelium in vivo, and the basis for such effects is unknown.
In the present study, we investigated the mechanisms underlying CRP actions on endothelium by testing the hypothesis that CRP attenuates eNOS activation in cultured endothelial cells. The resulting effect on monocyte adhesion was also determined. Because eNOS activation entails phosphatidylinositol 3-kinaseeCmediated increases in Ser1179 phosphorylation, which are counter-regulated by the phosphatase PP2A,16eC18 alterations in Ser1179 phosphorylation and the potential involvement of PP2A were investigated. To further identify the basis for this process, additional experiments tested the role of Fc receptors for IgG, which display high affinity for CRP and modulate CRP actions in immune response cells.19eC23 Moreover, studies of CRP-induced changes in endothelial function were performed in mice to delineate whether these mechanisms are operative in vivo.
Materials and Methods
Cell Culture and Transfection
Primary bovine aortic endothelial cells (BAEC) and human aortic endothelial cells (HAEC) (Cambrex Corp) were used within 7 passages. MFLM-4 were provided by Dr Ann Akeson (Children’s Hospital Medical Center, Cincinnati, Ohio). To study the role of FcRIIB, FcRII-negative COS-7 cells were transfected with human FcRIIB1 cDNA (a gift from Dr Catherine Sautes-Fridman, Paris, France),24 selected with Zeocin (Invitrogen), and cloned by limiting dilution. FcRIIB expression was tested by fluorescence-activated cell sorting (FACS) with the monoclonal antibody AT10 (provided by Dr P.M. Morganelli, White River Junction, Vermont). Zeocin-resistant cells not expressing detectable Fc receptor (FcR) served as controls.
eNOS Activation Assays
eNOS activation was assessed in whole cells by measuring [3H]L-arginine conversion to [3H]L-citrulline.25 Cell treatments included human recombinant CRP (Calbiochem), ascites-derived human CRP, purified and characterized as previously described,26 or human recombinant serum amyloid P component (SAP) (Calbiochem) added during a 15-minute preincubation and the 15-minute incubation for eNOS activation. For additional details, see the online data supplement available at http://circres.ahajournals.org. Control cells were exposed to CRP or SAP heated at 100°C for 60 minutes. Stimulated activity is expressed as percentage of basal activity, and results were confirmed in 3 experiments. eNOS activation was also evaluated ex vivo in isolated carotid arteries from 10- to 12-week-old male C57BL/6 mice by measuring cGMP accumulation during 2-minute incubations.27 The care and use of all study animals was approved by the International Animal Care and Use Committee at the University of Texas Southwestern Medical Center.
Monocyte Adhesion Assays
The adhesion of U937 cells to monolayers of BAEC was evaluated as previously described.28 Following U937 and endothelial cell coincubation and washing, cells were fixed and the number of adherent cells was counted. See the online data supplement for additional details.
Short-Interference RNA Preparation and Transfection
Double-stranded RNA with sequence 5'-CCAAGCUGCAAUCAUGGAA-3' was designed to target the open reading frame of the bovine PP2A catalytic subunit C29 (GenBank accession no. M16968). Scrambled sequence served as control. BAEC were transfected with 80 nmol/L RNA as described previously,30 and PP2A expression and eNOS activation were assessed 48 hours posttransfection.
Immunoblot Analyses
Immunoblots were performed to assess eNOS phosphorylation using antieCphospho-Ser1179 eNOS antibody (Cell Signaling Technology) and total eNOS abundance using eNOS monoclonal antibody (BD Biosciences Pharmingen).31 BAEC were starved overnight in the absence of serum or phenol red in DMEM before eNOS agonist treatment. Results shown were confirmed in 3 independent experiments.
RT-PCR for FcRIIA/B in Endothelium
FcRIIA/B expression was evaluated in HAEC by RT-PCR using Raji cells as positive controls. To assess and quantitate receptor expression in native endothelium, real-time RT-PCR studies were performed using RNA from endothelial cells isolated from the aorta, carotid artery, and heart of Tie2eCgreen fluorescent protein (GFP) transgenic mice by FACS analysis.32 See the online data supplement for additional details.
Carotid Artery Vascular Conductance in Mice
To determine whether CRP alters signaling to eNOS in vivo, Ach-induced changes in carotid artery vascular conductance were measured shortly before and after IP vehicle or CRP administration in mice.33 Studies were performed at 10 to 12 weeks of age in male C57BL/6 mice or in male FcRIIB+/+ versus FcRIIBeC/eC B6:129S mice (Jackson Laboratory).34 The dose of CRP used (250 e IP) yielded serum levels of 38±4 e/mL (n=6). See the online data supplement for additional details.
Statistical Analysis
Student t tests or ANOVA with NeumaneCKeuls post hoc testing were used to assess differences between 2 groups or among more than 2 groups, respectively, and significance was set at P<0.05.
Results
CRP Antagonism of eNOS Activation
To determine whether CRP alters eNOS activation, BAEC or HAEC were preincubated with heat-treated (control) or nontreated recombinant human CRP (5 e/mL), and eNOS stimulation was evaluated (Figure 1, left and right for BAEC and HAEC, respectively). Vascular endothelial growth factor (VEGF) stimulation of eNOS in either BAEC or HAEC was attenuated by CRP (Figure 1A). Similarly, CRP blunted eNOS activation by high-density lipoprotein (HDL) (Figure 1B) and by insulin (Figure 1C). Thus, CRP attenuates eNOS activation mediated by diverse agonists, intact CRP protein is required, and the process occurs in bovine and human endothelium.
Lipopolysaccharide (LPS) was not responsible for the observed effects on eNOS. Whereas CRP (5 e/mL) potently attenuated VEGF-stimulated eNOS activity, LPS (5 e/mL) had no effect (Figure 2A). In addition, human recombinant CRP and ascites-derived CRP26 caused comparable inhibition of eNOS activation (Figure 2B). SAP, the related pentraxin that is 60% homologous to CRP,35 also blocked eNOS activation (Figure 2C), and dose-response studies showed CRP effects at 5 e/mL and SAP effects at 2 e/mL (Figure 2D). These findings indicate that levels of CRP that have been associated with the risk of cardiovascular disease (3 to 10 e/mL)1 are operative and that the capacity to block eNOS activation is shared by CRP and SAP.
To determine whether CRP actions on eNOS alter endothelial cell function, the impact on monocyte adhesion was evaluated. LPS caused a marked increase in monocyte adhesion compared with control conditions (Figure 3A and 3B, respectively); this was reversed by the eNOS agonist insulin (Figure 3C), and the effect of insulin was confirmed to be NO dependent using NG-nitro-L-arginine methyl ester (L-NAME) (Figure 3D). CRP (3 e/mL) prevented the lessening of adhesion with insulin (Figure 3E), and this was not related to a change in eNOS expression (data not shown). The impact of CRP on adhesion was fully reversed by S-nitroso-N-acetyl-D, L-penicillamine (Figure 3F). Thus, CRP-induced declines in NO production promote monocyte adhesion to endothelium.
eNOS Phosphorylation and Involvement of PP2A
To define the mechanisms underlying CRP antagonism of eNOS, we first determined whether changes in gene transcription are involved. CRP caused comparable blockade of VEGF activation of eNOS in control and actinomycin DeCtreated cells (Figure 4A), indicating that CRP action is transcription independent. Because eNOS stimulation entails phosphatidylinositol 3-kinaseeCmediated increases in Ser1179 phosphorylation,17 changes in phosphorylation of the enzyme were investigated. Using HDL as agonist, an increase in phosphorylated eNOS occurred in control cells but not in CRP-treated cells (Figure 4B, top). Under these conditions, CRP exposure did not alter total eNOS abundance. The increase in eNOS phosphorylation by insulin was similarly blunted by CRP (Figure 4B, bottom), and comparable findings were obtained with estradiol-17 (10 nmol/L) (data not shown) or VEGF as agonist (Figure 4C). Because PP2A controls eNOS phosphorylation at Ser1179,16,18 the effect of the selective PP2A inhibitor okadaic acid (100 nmol/L) was investigated. In contrast to the diminution in eNOS phosphorylation observed with CRP alone, okadaic acid rescued Ser1179 phosphorylation in the presence of CRP (Figure 4C). In parallel, VEGF-stimulated eNOS activity was rescued by okadaic acid (Figure 4D). To confirm involvement of PP2A, short-interference RNA (siRNA) was used to diminish expression of the phosphatase in BAEC. With a 53% decline in PP2A protein expression (Figure 4E, inset), there was a parallel 48% rescue of eNOS activation by VEGF in the presence of CRP (Figure 4E). We also determined whether CRP blunts eNOS stimulation by bradykinin, which entails intracellular calcium elevation and eNOS phosphorylation.36 CRP inhibited bradykinin-induced eNOS phosphorylation and activation in an okadaic acideCsensitive manner (Figure 4F and 4G, respectively). These cumulative findings indicate that the negative modulation of eNOS by CRP is mediated by PP2A-induced changes in phosphorylation.
CRP Action In Vivo
In preparation for in vivo studies of CRP action in mice, the effect of CRP on Ach-mediated eNOS activation was evaluated in MFLM-4 mouse endothelial cells. eNOS stimulation by Ach was absent in CRP-treated cells (Figure 5A). CRP also caused blunted cGMP accumulation with Ach in isolated mouse carotid arteries (Figure 5B). In contrast, cGMP accumulation with the NO donor sodium nitroprusside was not altered by CRP (data not shown). Thus, CRP antagonizes Ach activation of eNOS ex vivo in mouse endothelium. Paralleling the findings with other agonists, CRP inhibited Ach-induced eNOS phosphorylation and activation in an okadaic acideCsensitive manner (Figure 5C and 5D, respectively). Thus, CRP blockade of signaling by Ach typifies the impact of CRP on multiple mediators of endothelial function.
To determine whether CRP attenuates eNOS activation in vivo, Ach-induced changes in carotid artery vascular conductance were measured in mice.33 Whereas the control vehicle had no effect (Figure 5E, left), following CRP administration the Ach response was blunted by 50% (Figure 5E, right). Thus, CRP actions on eNOS observed in cultured endothelium also occur in endothelium in vivo.
Role of FcRs
If the actions of CRP on eNOS require FcRs, which display high affinity for CRP and mediate its effects in immune response cells,1,19eC23 FcR crosslinking should yield the same phenotype as CRP. Aggregated IgG (aIgG) caused a concentration-dependent diminution in eNOS activation by VEGF (Figure 6A). In addition, aIgG blunted Ser1179 phosphorylation and eNOS activation in response to VEGF in an okadaic acideCsensitive manner (Figure 6B and 6C). These findings indicate that FcRs modify eNOS function via the activation of PP2A.
We then determined whether FcRIIs, the principal high-affinity receptors for CRP, are expressed in endothelium. In humans, FcRIIA is an activation receptor, and FcRIIB is an inhibitory receptor, and only FcRIIB has been identified in mice.37 In studies of HAEC, RT-PCR demonstrated mRNA expression for FcRIIB but not FcRIIA (Figure 6D). To determine whether FcRIIB is expressed in endothelium in vivo, endothelial cells were purified by FACS analysis from the aorta, carotid artery, and heart of Tie2-GFP transgenic mice.32 Following RNA isolation and amplification, quantitative real-time RT-PCR showed that the abundance of mRNA for eNOS, the target of interest for CRP action, was greater in endothelium from aorta and carotid artery compared with cardiac endothelium (Figure 6E, top). FcRIIB mRNA was detected in greater abundance in aortic and cardiac endothelium compared with carotid artery endothelium (Figure 6E, bottom). Thus, FcRIIB is expressed in human endothelial cells and in mouse endothelium, and the level of expression varies between blood vessel types.
The causal role of FcRIIB in CRP antagonism of eNOS was then tested in COS-7 cells expressing eNOS and scavenger receptor B type I (SR-BI) to enable eNOS activation by HDL. In control cells not expressing FcRIIB, CRP did not antagonize eNOS activation (Figure 7A, left). In contrast, in cells expressing FcRIIB, CRP blunted eNOS activation (Figure 7A, right), indicating that the action of CRP requires FcRIIB. CRP binding specifically to FcRIIB in this system was confirmed by FACS analysis (data not shown). The requirement for FcRIIB was also tested in vivo in comparisons of Ach-mediated increases in carotid vascular conductance in FcRIIB+/+ and FcRIIBeC/eC mice. Control vehicle did not alter Ach responses in either FcRIIB+/+ or FcRIIBeC/eC mice (Figure 7B), and Ach-induced increases in conductance were attenuated by CRP in FcRIIB+/+ mice (Figure 7C, left). In sharp contrast, CRP did not blunt Ach responses in FcRIIBeC/eC, and instead Ach-induced increases in conductance were enhanced by CRP (Figure 7C, right). These in vivo findings confirm that FcRIIB is required for CRP inhibition of eNOS. Interestingly, because there was actual enhancement of the Ach vasodilatory response by CRP in FcRIIBeC/eC mice, other mechanisms of CRP action may have been unmasked in the absence of FcRIIB. We postulate that the latter processes may involve stimulatory FcRs such as FcRIII, which cause increases in intracellular calcium on their activation and thereby would potentially enhance eNOS activity.37
Discussion
CRP levels are strongly correlated with increased risk for cardiovascular disease and with endothelial dysfunction related to decreased NO bioavailability.5eC12 However, the basis for potential effects of CRP on the endothelium has been unclear. Here we show that CRP causes potent antagonism of eNOS activation by diverse agonists resulting from changes in eNOS phosphorylation mediated by PP2A. Importantly, eNOS antagonism occurs at levels of CRP that have been associated with the risk of cardiovascular disease.1 When these processes are considered along with the previously known action of CRP to attenuate eNOS expression following prolonged exposure (24 hours),14,15 it is apparent that CRP decreases NO bioavailability by multiple mechanisms.
To link the effect of CRP on eNOS activation to a change in endothelial cell function, we show that CRP-induced declines in NO production underlie the promotion of monocyte adhesion by CRP in vitro. We also demonstrate in a mouse model that CRP antagonism of eNOS is operative in vivo. Such findings provide an explanation for the more than 50% decline in endothelium-dependent vasodilation that was recently observed following CRP infusion in hypercholesterolemic patients (E.S.G. Stroes, personal communication, 2005). Because there are multiple lines of evidence indicating that a loss in endothelial NO production plays a critical role in the pathogenesis of cardiovascular disease,38 we further propose that the resulting diminution in NO production may underlie the increased long-term cardiovascular risk associated with higher CRP levels in the absence of acute inflammation,8,39 as well as the poorer prognosis associated with even greater elevations in CRP during acute events.40 Whereas enhanced thrombosis has been effectively demonstrated in CRP transgenic mice,13 it is less clear from mouse models whether CRP accelerates atherogenesis.41eC43 Considering the complexity and diversity of cardiovascular diseases and their etiologies, further studies of the direct impact of eNOS-related CRP actions on vascular health and disease in animal models are now warranted.
In addition to the observed actions of CRP on eNOS, we found that the related pentraxin SAP had comparable effect at physiologic levels. SAP is a major acute-phase reactant in the mouse and a constitutive protein in the blood of humans, with basal concentrations of 2 to 10 and 40 e/mL, respectively.44,45 To date, SAP has not been associated with defects in vascular function. Although saturable binding of SAP to the IgG receptor subclass Fc, in particular FcRI, FcRIIa, and FcRIIIb, has been described in transfected COS cells,3 SAP may not be accessible to the endothelium in vivo because of association with various proteins in the plasma or vasculature.46eC48 More studies will be required to elucidate the role of SAP in vascular biology.
The known actions of CRP in immune-response cells are mediated by Fc receptors, with FcRII acting as the principal high-affinity receptor.19eC23 We demonstrate that human endothelial cells in culture and mouse endothelial cells in their native context express mRNA for FcRIIB. Attempts to detect FcRIIB protein in endothelium have been hindered by the lack of specificity of available antibodies and the low level of receptor abundance under quiescent conditions.49 However, using both gain-of-function and loss-of-function strategies in vitro and in vivo, we demonstrate that FcRIIB underlies the actions of CRP on vascular endothelium. Our studies are also the first to mechanistically link FcRIIB to PP2A in any paradigm, and the basis for the coupling of FcRIIB with PP2A warrants further investigation. Just as importantly, we show that the classical ligand for Fc receptors, aIgG, has identical action to CRP in endothelium. It is well established that patients with rheumatoid arthritis and systemic lupus erythematosus have a higher incidence of cardiovascular disease and endothelial dysfunction that is not explained by traditional risk factors.50eC54 We propose that elevated levels of CRP or circulating immune complexes engage endothelial FcRIIB to attenuate eNOS activity in these patients, thereby adversely affecting endothelial function and possibly contributing to their greater cardiovascular disease risk.
Collectively the present observations reveal a novel series of mechanisms by which CRP is a direct mediator of endothelial dysfunction. Our findings provide a new framework for understanding how CRP, and also circulating immune complexes, may contribute to the pathogenesis of vascular disease. It is anticipated that further research in this realm will lead to new prophylactic and therapeutic strategies to combat the vascular complications of multiple inflammatory and autoimmune disorders
Acknowledgments
This work was supported by NIH grants HL75473 (to P.W.S.), HL06296 (to G.D.T.), and AG02467 (to D.S.). The project was also supported by American Heart Association Scientist Development Award 0235107N (to C.M.), the Children’s Medical Center Dallas Foundation (to A.K.G.), the Crystal Charity Ball Center for Pediatric Critical Care Research and the Lowe Foundation (to P.W.S.), and the Simmons Family Foundation (to D.R.K.). We are indebted to Miranda King for technical assistance and to Marilyn Dixon for assistance in preparing the manuscript.
Both authors contributed equally to this study.
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