Cardiovascular Research

Cardiovascular Research 2006 70(1):31-41; doi:10.1016/j.cardiores.2006.01.025

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Copyright © 2006, European Society of Cardiology

Circulating cardiovascular disease risk factors and signaling in endothelial cell caveolae

Chieko Mineo and Philip W. Shaul^*

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd, Dallas, TX, USA

^* Corresponding author. Tel.: +1 214 648 2015; fax: +1 214 648 2481. Email address: philip.shaul{at}utsouthwestern.edu

Received 13 December 2005; revised 9 January 2006; accepted 13 January 2006

	Abstract

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
Caveolae are a subset of lipid rafts that are prevalent on the plasma membrane of endothelial cells. They compartmentalize signal transduction molecules which regulate multiple endothelial functions including the production of nitric oxide (NO) by the caveolae resident enzyme endothelial NO synthase (eNOS). Recent studies have demonstrated that circulating factors known to modify cardiovascular disease risk regulate signaling in endothelial cell caveolae. In particular, high density lipoprotein (HDL) maintains the lipid environment in caveolae, thereby promoting the retention and function of eNOS in the domain, and it also causes direct activation of eNOS via scavenger receptor type BI (SR-BI)-induced kinase signaling. Estrogen binding to estrogen receptors (ER) in caveolae also activates eNOS, and this occurs through G protein and kinase activation. Discrete domains within SR-BI and ER mediating signal initiation in caveolae have been identified. Counteracting the promodulatory actions of HDL and estrogen, C-reactive protein (CRP) antagonizes eNOS through FcRIIB and PP2A, which dephosphorylates and inactivates the enzyme. The endothelial cell functions modified by these processes include the regulation of monocyte adhesion and endothelial cell migration. Thus, signaling events in caveolae invoked by known circulating cardiovascular disease risk factors have major impact on endothelial cell phenotypes of importance to cardiovascular health and disease.

KEYWORDS Caveolae; C-reactive protein; Endothelium; Estrogen; High density lipoprotein; Nitric oxide

	1. Introduction

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
The endothelial cell is critically involved in neovascularization and in the regulation of the structure and function of established blood vessels. Endothelial cells generate signaling molecules such as nitric oxide (NO), prostacyclin and endothelin which serve diverse autocrine and paracrine functions, and they form a monolayer which modulates local hemostasis and thrombolysis and provides a non-permeable barrier protecting the underlying vascular smooth muscle from circulating growth-promoting factors [1–4]. As the guardian of the vascular wall, the endothelium is the "first-responder" to multiple physical, biochemical and cellular events occurring in the lumen.

Over the past several years our knowledge of endothelial cell responses to external stimuli has been enhanced by the identification and study of signaling molecules and their interactions in caveolae, which are a specialized subset of lipid rafts that are prevalent on the endothelial cell plasma membrane. Caveolae are enriched in cholesterol, glycosphingolipids, sphingomyelin and lipid-anchored membrane proteins. This review will highlight recent advances in our understanding of endothelial cell signaling events in caveolae, with a focus on mechanisms regulating the activity of the resident enzyme endothelial NO synthase (eNOS). Emphasis is placed on processes which provide mechanistic coupling between circulating factors known to modify cardiovascular disease risk and their abilities to directly govern endothelial function. After reviewing the mechanisms underlying eNOS localization in caveolae, the capacity of high density lipoprotein (HDL) cholesterol to activate eNOS will be discussed. The promodulatory actions of estrogen on eNOS will also be outlined. Representing the more recent consideration of disease-related eNOS antagonism, inhibitory actions of C-reactive protein (CRP) will be reviewed. Finally, key questions guiding the future direction of research in this field will be highlighted.

	2. eNOS localization in endothelial cell caveolae

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
eNOS is one of the three isoforms of NOS which generates NO upon the conversion of L-arginine to L-citrulline. eNOS is activated by increases in intracellular calcium and by changes in phosphorylation mediated by upstream kinases. In particular, with many agonists receptor tyrosine kinases (TK) or nonreceptor TK activate PI3 kinase/Akt kinase to stimulate eNOS enzymatic activity by causing the phosphorylation of Ser-1179. In certain contexts there are also alterations in Thr-497 phosphorylation, which attenuates enzyme activity. The NO produced prevents thrombosis, adhesion molecule expression and apoptosis, and it promotes endothelial cell growth and migration and vasodilation while attenuating vascular smooth muscle growth and migration. Diminished NO production or bioavailability has been implicated in the pathogenesis of systemic and pulmonary hypertension and in other vascular disorders including atherosclerosis [1,2,5–9].

Since eNOS activity is regulated by diverse extracellular stimuli and the NO produced is a labile, cytotoxic molecule with paracrine functions [5,6], the intracellular site of NO synthesis has a major influence on the biological impact of the enzyme. eNOS is primarily associated with caveolae [10,11], and optimal targeting of the enzyme to caveolae requires N-terminal myristoylation and palmitoylation [10]. In addition, the status of cholesterol in caveolae is critical to normal eNOS function [12]. Oxidized LDL (oxLDL) causes depletion of caveolae cholesterol in cultured endothelium via the scavenger receptor CD36, leading to eNOS redistribution away from the plasma membrane and a diminished capacity to activate the enzyme [12,13]. Paralleling the findings in cell culture, Ach administration to wild-type mice with eNOS localized in caveolae results in a fall in blood pressure, whereas Ach does not alter blood pressure in hypercholesterolemic apoE null mice in which eNOS is not present in caveolae. In contrast, normal eNOS localization in caveolae and normal Ach-induced blood pressure responses occur in apoE/CD36 double knockout mice [14]. Thus, pathologic lipoprotein and cholesterol status disrupts eNOS subcellular localization to caveolae and thereby attenuates the function of the enzyme. This mechanism may be operative in the early stages of hypercholesterolemia-induced vascular disease, when there is impairment of eNOS responses to receptor-dependent stimuli [1,2,7].

Counteracting caveolae cholesterol depletion by oxLDL via CD36, HDL maintains caveolae cholesterol content, retains eNOS in the domain, and preserves Ach-induced activation. This process is not related to the inhibition of cholesterol removal from caveolae by oxLDL; instead, it is due to the provision of cholesterol esters by HDL [13]. Moreover, scavenger receptor class B Type I (SR-BI), the high affinity receptor for HDL, is in endothelial caveolae, and it mediates the ability of HDL to reverse the impact of oxLDL on eNOS localization and function [13]. Thus, in the presence of oxLDL, the HDL/SR-BI tandem preserves the lipid environment within caveolae, thereby maintaining normal eNOS localization and function and possibly explaining a portion of the antiatherogenic properties of HDL.

	3. HDL activation of eNOS

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
In addition to modifying eNOS localization when the lipid environment in caveolae is overtly altered, studies of cultured endothelial cells have shown that HDL is a potent agonist of eNOS. Further experiments in cultured cells showed that SR-BI mediates eNOS activation by HDL. In parallel, HDL causes endothelium- and NO-dependent relaxation of aortic rings from wild-type but not SR-BI null mice (Fig. 1A, B). In addition, functional coupling of SR-BI to eNOS is demonstrable in isolated endothelial cell caveolae (Fig. 1C) [15]. These mechanisms may explain the positive relationship observed between circulating HDL levels and endothelium-dependent vasodilation [16–21].

View larger version (28K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 HDL initiates endothelial cell signaling via SR-BI in caveolae. (A) HDL stimulates vasodilation through eNOS activation. Following precontraction of rings of thoracic aortae from wild-type mice with phenylephrine (arrow), responses to control buffer (CON), HDL at 10µg/ml (open arrowhead) or 25µg/ml (closed arrowhead), HDL with prior L-NAME treatment, or HDL with endothelium-denuded (-endo) rings were evaluated. (B) SR-BI is required for HDL-induced vasodilation. The effects of control buffer (CON) or HDL were tested in phenylephrine precontracted rings of thoracic aortae from SR-BI+/+ or SR-BI– / – mice. (C) HDL stimulates eNOS in isolated caveolae membranes. [3H]L-arginine conversion to [3H]L-citrulline was measured in noncaveolae and caveolae membranes from endothelial cells during 60-min incubations performed in the absence (basal, ‘B’) or presence of HDL or LDL. In (B and C), values are mean±S.E.M., n=4–6, *P<0.05 versus control or basal. (D–F) SR-BI modulates reendothelialization in mouse carotid artery. Five days after thermal injury, the area of remaining denudation was evaluated in arteries from SR-BI+/+ (D) and SR-BI– / – mice (E) by Evan's blue dye incorporation. (F) Area of denudation was quantified and is expressed in arbitrary units. Values are mean±S.E.M., n=7–8 mice/group. *P<0.05 versus SR-BI+/+. Reprinted with permission [15,28].

 
ApoA-I is the apolipoprotein principally responsible for the atheroprotective features of HDL [22]. In cultured endothelial cells, apoA-I–eNOS interaction and perinuclear colocalization have been reported [23]. However, lipid-free apoA-I does not activate eNOS, yet anti-apoA-I antibody blocks eNOS activation by HDL in isolated endothelial cell plasma membranes [15]. Thus, apoA-I is necessary but not sufficient for eNOS stimulation.

The signal transduction events underlying HDL activation of eNOS have been elucidated. HDL causes eNOS phosphorylation at Ser-1179, and PI3 kinase inhibition or dominant negative Akt inhibits both HDL-mediated eNOS phosphorylation and activation. Further studies have shown that Src family kinases are upstream of PI3 kinase-Akt in the HDL signaling pathway. In addition, HDL activates MAP kinase through PI3 kinase, and MAP kinase/extracellular signal-regulated kinase kinase inhibition attenuates eNOS stimulation by HDL without affecting eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt does not alter HDL-induced MAP kinase activation. Thus, HDL stimulates eNOS through Src activation, which leads to parallel activation of Akt and MAP kinases and their independent modulation of the enzyme [24]. In addition, Ca²⁺ plays an important role [25].

The involvement of lysophospholipids associated with HDL in eNOS activation has been suggested. Sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P) and lysosulfatide (LSF) are all constituents of native HDL, and HDL, SPC, S1P, and LSF cause similar eNOS-dependent relaxation of precontracted aortic rings from mice. Experiments with aortas from mice lacking the lysophospholipid receptor S1P₃ indicate that 50–60% of the response to native HDL is mediated by lysophospholipids [25]. However, the intravenous administration of HDL stimulates myocardial perfusion in vivo equivalently in wild-type and S1P₃ null mice but not in eNOS null mice [26]. As such, the role of eNOS in HDL-induced vascular responses remains clear whereas the involvement of lysophospholipids is less certain. It has also been reported that HDL from females has greater capacity to stimulate eNOS than does HDL from males, with further experiments suggesting that HDL-associated estradiol is involved [27], but this has not been a consistent observation [25].

HDL also induces other important endothelial cell responses which are independent of NO. In particular, HDL stimulates endothelial cell migration in vitro via SR-BI-mediated activation of Rac GTPase [28]. This process requires the apoA-I, phospholipids and cholesterol components of HDL, and it occurs through the activation of Src kinases, PI3-kinase, and p44/42 MAP kinases [28]. Additional studies indicate involvement of a G protein-dependent mechanism mediated by S1P [29]. In parallel, carotid artery reendothelialization after perivascular electric injury is blunted in apoA-I null mice, and the reconstitution of apoA-I expression in apoA-I null mice rescues reendothelialization. Furthermore, there is impaired reendothelialization in SR-BI null mice (Fig. 1D–F) [28]. Thus, in the context of all in vivo factors governing endothelial cell phenotype, HDL and SR-BI are potent modulators of endothelial cell migration.

The molecular basis of HDL signaling has been investigated. In cultured endothelium, short-term exposure to HDL or methyl-β-cyclodextrin causes equivalent eNOS stimulation, whereas cholesterol-loaded methyl-β-cyclodextrin does not. Cholesterol-free Lp2A-I particles containing lipid-free apoA-I and phosphatidylcholine also activate eNOS, whereas cholesterol containing Lp2A-I particles do not. In addition, phosphatidylcholine-loaded HDL causes greater stimulation of eNOS than native HDL, and blocking antibody to SR-BI, which retards cholesterol efflux, prevents eNOS activation by HDL. Furthermore, heterologous expression experiments using COS-M6 cells have revealed that wild-type SR-BI mediates eNOS activation by both HDL and small unilamellar vesicles, whereas the SR-BI mutant AVI, which is incapable of efflux to small unilamellar vesicles [30], signals only in response to HDL. Moreover, eNOS activation by both HDL and methyl-β-cyclodextrin is SR-BI-dependent [31]. These findings indicate that signaling by HDL requires cholesterol flux, that the apolipoprotein and phospholipid components of HDL are sufficient to induce signal, and that SR-BI functions as a plasma membrane cholesterol sensor.

The features of SR-BI required for signaling have also been elucidated. Using a splice variant of SR-BI, SR-BII, and mutant and chimeric class B scavenger receptors, it was determined that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains are required. Since these domains are not involved in regulating cholesterol flux, cholesterol movement alone is not sufficient to initiate signaling. Further studies showed that cell cholesterol binds directly to the C-terminal transmembrane domain of SR-BI [31]. This is a characteristic of other cholesterol sensing proteins such as SCAP, which undergoes a conformational change with an alteration in cholesterol binding that modifies its function [32]. The requirement for the C-terminal PDZ-interacting domain suggests additional involvement of an adaptor protein, which may promote interactions between SR-BI and downstream signaling molecules. Although these underlying mechanisms are yet to be tested for SR-BI, they may be critical to signaling in caveolae in response to changes in membrane cholesterol.

	4. Estrogen modulation of eNOS

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
There is considerable evidence that the hormone estrogen has potent direct actions on vascular cells including endothelium [33,34]. These processes may underlie the lower susceptibility to vascular disease in premenopausal females compared with males and the cardiovascular benefits of estrogen replacement therapy when provided under certain circumstances [35]. Many of the vascular actions of estrogen are mediated by increases in bioavailable NO. Whereas a portion of this effect relates to the upregulation of eNOS expression, an important contribution is derived from the nongenomic activation of eNOS by estrogen [36]. The basis for nongenomic effects of estradiol (E₂) on eNOS function and the role of estrogen receptors (ER), which classically serve as transcription factors, was first elucidated in cultured endothelial cells. It was initially shown that E₂ acutely (5–10min) stimulates eNOS activity, that the response is attenuated by ER antagonism but not by inhibition of gene transcription, that ER is expressed in endothelium, and that ER overexpression in endothelial cells causes enhancement of the acute response to E₂ that is blocked by ER antagonism (Fig. 2A). It was also observed in COS-7 cells that the coexpression of ER with eNOS yields cells in which the acute stimulation of eNOS by E₂ is demonstrable [37–39]. Collectively these findings revealed that ER mediate the nongenomic activation of eNOS.

View larger version (30K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Estrogen stimulates eNOS through an ER-G protein complex in caveolae. (A) Estradiol (E2) stimulates eNOS activity via ER. 72h following transfection with sham plasmid or ER cDNA, [3H]L-arginine conversion to [3H]L-citrulline was measured in intact endothelial cells over 15min in the absence or presence of 10– 8M E2, with or without 10– 5 M ICI 182,780 added simultaneously. Values are mean±S.E.M.; n=4–6. *P<0.05 vs. basal, P<0.05 vs. sham. (B) Localization of ER in endothelial cell caveolae. Immunoblot analysis was performed for ER and caveolin-1 in noncaveolae membranes (NCM) and caveolae membranes (CAV) obtained from endothelial cell plasma membranes. (C) E2-mediated activation of eNOS occurs in endothelial cell caveolae membranes. [3H]-L-arginine conversion to [3H]-L-citrulline was measured in noncaveolae and caveolae membranes obtained from endothelial cell plasma membranes. Incubations were performed over 60min in the absence (basal, B) or presence of 10– 8super M E2 with or without 10– 5super M ICI 182,780 added. NOS activity was undetectable in noncaveolae fractions in all groups, and it was also not detected in caveolae under basal conditions. Values are mean±S.E.M., n=4 to 6. *P<0.05 vs. basal; P<0.05 vs. E2 alone. (D) E2 activation of eNOS is blunted by pertussis toxin (PT). Intact endothelial cells were pretreated with vehicle or 100ng/ml PT for 120min, and [3H]-L-arginine conversion to [3H]-L-citrulline was assessed under basal conditions (B) or in the presence of 10– 5M acetylcholine (Ach) or 10– 8M E2 over 15min in the continued presence of vehicle or PT. Values are mean±S.E.M.; n=3. *P<0.05 vs. basal, P<0.05 vs. no PT. (E) Coimmunoprecipitation of ER and Gi from endothelial cell plasma membranes. Cells were treated with vehicle or 10– 8M E2 for 20min, plasma membranes were isolated, immunoprecipitation was done with ER antibody, and immunoblot analyses were performed for ER and Gi. (F) Specific domains of ER mediate Src phosphorylation. COS-7 cells were transiently transfected with cDNA for either wild-type ER, ER250–274, or ER185–251. 48h later the cells were treated with 10– 8M E2 for 0–15min, and cell lysates were obtained and analyzed by immunoblot using anti phospho-Tyr416 Src polyclonal antibody (pSrc) or anti-Src monoclonal antibody to assess relative Src phosphorylation. (G) Specific domain of ER mediates PI3 kinase association. Postnuclear supernatants were obtained after E2 treatment and the interaction between ER and the p85 subunit of PI3 kinase was assessed by coimmunoprecipitation and immunoblot analyses for p85 and ER. In (F and G) values are mean±S.E.M., n=3, *P<0.05 vs. basal. Reprinted with permission [39,46,51,53].

 
The signal transduction mechanisms by which E₂ stimulates eNOS have also been delineated. Multiple lines of evidence support important roles for tyrosine kinases including Src family kinases, MAP kinases, and PI3 kinase/Akt kinase, and direct interaction between ER and the p85 subunit of PI3-kinase has been demonstrated [39–42]. In the two initial reports of eNOS stimulation in cultured endothelial cells, it was unclear if calcium is also involved. It was noted in studies in ovine endothelial cells that the removal of extracellular calcium completely prevented the response [37], yet changes in cytosolic calcium levels were not detected in parallel with eNOS activation by E₂ in HUVEC [38]. Later work in bovine and human endothelial cells indicated that there is a transient rise in intracellular calcium concentration upon E₂ exposure [43,44]. Furthermore, both E₂-induced Akt activation and eNOS translocation from the plasma membrane are calcium-dependent [42,43]. Thus, E₂ activation of eNOS is most likely a calcium-dependent process, but global increases in intracellular calcium may not be required.

Additional mechanisms of eNOS regulation by E₂ have been identified. In studies of HUVEC, inhibitors of Hsp90 function prevented E₂-stimulated NO release and cGMP production. E₂ also was found to induce Hsp90–eNOS association, and this event was prevented by ER antagonism, providing further evidence for a role for Hsp90 [45]. Thus, the short-term effects of estrogen on eNOS which are central to cardiovascular physiology are mediated by ER functioning nongenomically, and multiple signal transduction events are likely to be involved.

Our understanding of nongenomic E₂ activation of eNOS was further enhanced by elucidation of the subcellular site of interaction between ER and eNOS. It was found that E₂ activates eNOS in isolated endothelial cell plasma membranes and that the response is inhibited by ER antagonism. Immunoidentification experiments with different antibodies to ER detected a single 67-kDa protein species in plasma membrane that is identical in size to the protein detected in nucleus and cytosol. In addition, epitope-tagged ER (ER-myc) expressed in COS-7 cells is directed to the plasma membrane. These cumulative findings indicate that E₂-stimulated eNOS activity is mediated by plasma membrane-associated ER [46]. Investigations using membrane-impermeant E₂ conjugated to bovine serum albumin provided additional evidence of eNOS signaling by cell surface ER [40,44]. Furthermore, ER protein and functional coupling to eNOS is demonstrable in isolated endothelial caveolae (Fig. 2B, C) [46]. Splice variants of ER may also participate in the nongenomic activation of eNOS. In particular, a 46-kDa form with functional coupling to eNOS has been identified [47]. A variety of mechanisms have been proposed to underlie ER localization to caveolae. Palmitoylation of ER occurs at Cys 447, and palmitoylation enhances membrane localization and the capacity for signal initiation [47,48]. In addition, within the ER ligand binding domain there is a caveolin-1 interaction site which promotes targeting to caveolae [49].

Along with ER, ERβ expression has been demonstrated in endothelial cells [33]. In cultured endothelium with constitutive ERβ expression, a subpopulation of ERβ is localized to plasma membrane, ERβ overexpression enhances eNOS stimulation by E₂, and the response to E₂ is inhibited by the ERβ-selective antagonist RR-tetrahydrochrysene (THC). In addition, eNOS activation through ERβ can be reconstituted in COS-7 cells. Furthermore, ERβ protein is detected and THC attenuates E₂ stimulation of eNOS in isolated endothelial cell caveolae [50]. Thus, both ER and ERβ have nongenomic action in endothelial cell caveolae to regulate eNOS activity.

Evidence of G protein involvement in ER coupling to eNOS first came from the observation that E₂ activation of the enzyme is pertussis toxin (Ptox)-sensitive (Fig. 2D). Coimmunoprecipitation studies of plasma membranes from COS-7 cells transfected with ER and specific G proteins demonstrated E₂-stimulated interaction between ER and Gi but not between ER and either Gq or Gs. Importantly, the ER–Gi interaction is blocked by ER antagonism and Ptox, and it is demonstrable in endothelial cell plasma membranes (Fig. 2E). Furthermore, the cotransfection of Gi into COS-7 cells expressing ER and eNOS yields an increase in E₂-mediated eNOS stimulation, whereas cotransfection with a protein regulator of G protein signaling, RGS4, inhibits the E₂ response. These findings indicate that eNOS stimulation by E₂ requires plasma membrane ER coupling to Gi [51]. This coupling may be facilitated by the scaffolding protein striatin [52]. Thus, novel G protein coupling enables ER to initiate signal transduction in caveolae.

The features of ER mediating nongenomic signaling to eNOS have been elucidated recently. In crosslinking experiments it was demonstrated that E₂ causes plasma membrane-associated ER to form dimers. However, eNOS activation by E₂ is unaltered for a dimerization-deficient mutant ER (ERL511R). In contrast, ER mutants lacking the nuclear localization signals (NLS), NLS2,3 (ER250–274) or the DNA binding domain (ER185–251), which target normally to caveolae, are incapable of activating eNOS. The loss of NLS2/NLS3 prevents Src and p42/44 MAPK activation, and it alters ligand-induced PI3 kinase-ER interaction and prevents eNOS phosphorylation (Fig. 2F, G). The loss of the DNA binding domain does not change E₂ activation of Src or p42/44 MAPK, but ligand-induced PI3 kinase-ER binding and eNOS phosphorylation does not occur. Thus, domains of ER previously known to modulate its genomic actions are involved in signaling on the plasma membrane [53].

	5. CRP modulation of eNOS

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
C-reactive protein (CRP) is an acute phase reactant which is positively correlated with cardiovascular disease risk and endothelial dysfunction [54–60]. In recent work in bovine and human aortic endothelial cells in culture, it was found that brief treatment with recombinant human CRP fully attenuates eNOS activation by HDL, E₂, vascular endothelial growth factor (VEGF) and insulin (Fig. 3A). Serum amyloid P component (SAP), the related pentraxin which is 60% homologous to CRP [61], also blocks eNOS activation, and dose–response studies show CRP effects at 5µg/ml and SAP effects at 2µg/ml. CRP also blunts cGMP accumulation with Ach in isolated mouse carotid arteries. Studies of monocyte adhesion further demonstrate that CRP alters endothelial cell functions mediated by NO. Whereas LPS-induced monocyte adhesion is blunted by eNOS agonists such as insulin, CRP prevents the lessening of adhesion with eNOS activation, and the impact of CRP is reversed by NO donors (Fig. 3B). Comparable observations have been made in studies of CRP and HDL- or E₂-mediated antagonism of monocyte adhesion [62]. Thus, levels of CRP associated with the risk of cardiovascular disease (3–10µg/ml) [60] cause antagonism of eNOS activation and resulting impairment of NO-dependent function in endothelial cells.

View larger version (44K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 C-reactive protein inhibits eNOS activation via FcRIIB in endothelium. (A) CRP inhibits eNOS activation. Endothelial cells were exposed to heat-treated (control) or active CRP (5µg/ml for 15min) and eNOS stimulation by insulin (500nM) was evaluated. Values are mean±S.E.M., n=4–6, *p<0.05 vs. basal, p<0.05 vs. control. (B) CRP antagonism of eNOS promotes monocyte adhesion. Endothelial cells were treated for 18h with media alone (control), lipopolysaccharide (LPS, 100ng/ml), LPS+insulin (Ins., 500nM), LPS+Ins.+L-NAME (2mM), LPS+Ins.+CRP (3µg/ml), or LPS+Ins.+CRP+the NO donor S-nitroso-N-aceyl-D,L,-penicillamine (SNAP, 20µM). The adhesion of the monocyte cell line (U937) was then evaluated. Images of cobblestone-shaped endothelial cells and round monocytes are representative optical fields. (C) CRP inhibits eNOS phosphorylation through PP2A activation. Endothelial cells were preincubated in the absence or presence of CRP (5µg/ml), okadaic acid (OK, 100nM), or CRP plus OK, treated with VEGF (100ng/ml for 0–10min), and lysates were analyzed for phospho-eNOS (peNOS) or total eNOS. (D) CRP inhibits eNOS activation via PP2A activation. Endothelial cells were preincubated in the absence or presence of OK, CRP, or OK and CRP, and eNOS activation by VEGF was assessed in the continued absence or presence of OA and/or CRP. Values are mean±S.E.M., n=4–6, *p<0.05 vs. basal, p<0.05 vs. no OK. (E) CRP antagonism of eNOS is mediated by FcRIIB. COS-7 cells expressing eNOS and SR-BI (control, left panel), or eNOS, SR-BI and FcRIIB (FcRIIB, right panel) were preincubated with buffer alone or buffer plus 5µg/ml CRP, and eNOS activation by 10µg/ml HDL was assessed in the continued absence or presence of CRP. Values are mean±S.E.M., n=4–6, *p<0.05 vs. basal, p<0.05 vs. no CRP. (F) FcRIIB mediates CRP antagonism of eNOS in vivo. FcRIIB+/+ or FcRIIB– / – mice were instrumented and increases in carotid artery conductance with acetylcholine (Ach) were measured before and after CRP administration. Dose–responses to Ach were determined sequentially at baseline (•), 60min after CRP was injected intraperitoneally (), and 10min after the administration of L-NAME (). Values are mean±S.E.M., n=7 mice/group, *p<0.05 vs. baseline. Reprinted with permission [62].

 
The mechanisms underlying CRP antagonism of eNOS have also been elucidated. CRP causes comparable blockade of eNOS activation in control and actinomycin D-treated cells, indicating that CRP action is transcription-independent. The impairment in eNOS activation is due to diminished agonist-induced eNOS phosphorylation at Ser-1179. Further studies using okadaic acid and siRNA demonstrated that both the diminution in Ser-1179 phosphorylation and the loss of eNOS activation caused by CRP are due to the activation of the phosphatase PP2A (Fig. 3C, D) [62], which controls eNOS phosphorylation at Ser- 1179 [63,64].

The role of Fc receptors, which display high affinity for CRP and mediate its effects in immune response cells [65–69], has also been evaluated. Paralleling the actions of CRP, aggregated IgG (aIgG), the known ligand for Fc receptors, causes a diminution in eNOS activation in cultured endothelial cells. In addition, aIgG blunts eNOS Ser-1179 phosphorylation, and all of these effects of aIgG are okadaic acid-sensitive, indicating that Fc receptors modify eNOS function via PP2A. Consistent with this conclusion, the inhibitory Fc receptor FcRIIB is expressed in human endothelial cells in culture and in mouse endothelium in vivo [62]. The causal role of FcRIIB in CRP antagonism of eNOS is evident from experiments in COS-7 cells expressing eNOS and SR-BI to enable eNOS activation by HDL. In control cells not expressing FcRIIB, CRP does not antagonize eNOS activation; in contrast, in cells expressing FcRIIB, CRP blunts eNOS activation (Fig. 3E). Thus, FcRIIB is expressed in endothelium, and the receptor mediates the actions of CRP on eNOS [62]. In B cells, FcRIIB is localized to lipid rafts [70]. Similar caveolae/raft localization of FcRIIB in endothelial cells is yet to be demonstrated.

The capacity of CRP to attenuate eNOS activation via FcRIIB has also been demonstrated in vivo in studies of Ach-induced increases in carotid artery vascular conductance in mice. Following CRP administration, the Ach response in wild-type FcRIIB^+/+ mice is blunted by 50%. In contrast, CRP does not blunt Ach responses in FcRIIB^{– / –}, and instead Ach-induced increases in conductance are enhanced by CRP (Fig. 3F) [62]. Interestingly, since there is enhancement of the Ach vasodilatory response by CRP in FcRIIB^{– / –}, other mechanisms of CRP action may have been unmasked in the absence of FcRIIB. The latter processes may involve stimulatory FcR such as FcRIII, which increases intracellular calcium when activated and thereby would potentially enhance eNOS activity [71].

	6. Summary and future direction

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
It is now evident that multiple signaling pathways relevant to circulating cardiovascular disease risk factors converge in caveolae to regulate eNOS. The localization of the enzyme in caveolae is critical to normal NO production activated by various agonists, and in hypercholesterolemic mice eNOS loss from caveolae and resultant dysfunction have been demonstrated. Work in cell culture further indicates that HDL and SR-BI play important roles in maintaining normal eNOS localization via modulation of the lipid environment in caveolae. HDL also directly activates eNOS through SR-BI, which is colocalized with the enzyme in caveolae (Fig. 4). HDL-mediated cholesterol efflux triggers the activation of multiple kinases and induces eNOS phosphorylation, and these processes are dependent on specific domains of SR-BI. Estrogen binding to ER in caveolae also stimulates eNOS through G proteins and the activation of kinases (Fig. 4). Multiple domains of ER demonstrate distinctive roles in mediating membrane initiated signaling, and protein–protein interactions between ER and other caveolae proteins are also involved. In contrast to HDL and estrogen, CRP antagonizes eNOS through FcRIIB (Fig. 4). The effect of CRP is mediated by PP2A, which dephosphorylates eNOS, resulting in the inactivation of the enzyme. The endothelial cell functions modified by these processes include the regulation of monocyte adhesion and endothelial cell migration, and consequently these signaling events in caveolae have profound implications on cardiovascular health and disease.

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Signaling molecules in caveolae governing endothelial cell phenotype. HDL binding to SR-BI in caveolae activates Src family kinases, PI3 kinase/Akt kinase and p42/44 MAP kinases, leading to eNOS activation and the inhibition of monocyte adhesion. Through NO-independent processes, signaling induced by HDL also promotes endothelial cell migration. Estradiol (E2) binding to caveolae-associated ER and ERβ also activates Src family kinases, PI3 kinase/Akt kinase and p42/44 MAP kinases, resulting in eNOS activation, the inhibition of monocyte adhesion, and the stimulation of endothelial cell migration. The actions of membrane-associated ER are mediated by heterotrimeric G proteins. In contrast to the promodulatory actions of HDL and E2, CRP binding to FcRIIB antagonizes eNOS activation through PP2A-induced dephosphorylation of the enzyme, leading to blunted endothelial cell migration and greater monocyte adhesion. It is important to note (*) that although FcRIIB localization to caveola/rafts has been demonstrated in immune response cells, comparable localization in endothelial cells is yet to be confirmed.

 
The current questions in this area of research are numerous. The mechanisms by which HDL-induced cholesterol flux transduces signal to downstream effectors are completely unknown. The specific roles of the C-terminal transmembrane and cytoplasmic domains of SR-BI in signaling initiated upon cholesterol sensing by the receptor also warrant further study. The processes underlying caveolae-associated ER coupling to G proteins are currently unknown, and additional adaptor and partner signaling molecules for membrane ER await discovery. The basis for CRP binding to FcRIIB and for FcRIIB coupling to PP2A remain to be elucidated. Although FcRIIB localization to caveolae/rafts has been demonstrated in immune response cells, comparable localization and the mechanisms regulating such targeting in endothelial cells are yet to be revealed. In addition, the nuclear responses to HDL, estrogen and CRP signaling in caveolae are currently unknown. As importantly, other signaling modules in caveolae governing endothelial cell phenotype are likely to exist and need to be identified. Moreover, the developmental and disease-related implications of these processes await further study in intact animal models. With continued efforts to elucidate the molecular components and signaling events occurring in endothelial cell caveolae, it is expected that valuable new lessons will be learned from the guardian cell of the vascular wall to ultimately yield new strategies to prevent and treat vascular disease.

	Acknowledgments

Top Abstract 1. Introduction 2. eNOS localization in... 3. HDL activation of... 4. Estrogen modulation of... 5. CRP modulation of... 6. Summary and future... Acknowledgments References

 
This work was supported by NIH grants HD30276, HL58888 and HL75473 (PWS), American Heart Association grant 0235107N (CM), the Crystal Charity Ball Center for Pediatric Critical Care Research, and the Lowe Foundation.

	Notes

 
Time for primary review 21 days

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