Supplementation With the Sialic Acid Precursor N-Acetyl-D-Mannosamine Breaks the Link Between Obesity and Hypertension.

BACKGROUND: Obesity-related hypertension is a common disorder, and attempts to combat the underlying obesity are often unsuccessful. We previously revealed that mice globally deficient in the inhibitory immunoglobulin G (IgG) receptor FcγRIIB are protected from obesity-induced hypertension. However, how FcγRIIB participates is unknown. Studies were designed to determine if alterations in IgG contribute to the pathogenesis of obesity-induced hypertension. METHODS: Involvement of IgG was studied using IgG µ heavy chain-null mice deficient in mature B cells and by IgG transfer. Participation of FcγRIIB was interrogated in mice with global or endothelial cell-specific deletion of the receptor. Obesity was induced by high-fat diet (HFD), and blood pressure (BP) was measured by radiotelemetry or tail cuff. The relative sialylation of the Fc glycan on mouse IgG, which influences IgG activation of Fc receptors, was evaluated by Sambucus nigra lectin blotting. Effects of IgG on endothelial NO synthase were assessed in human aortic endothelial cells. IgG Fc glycan sialylation was interrogated in 3442 human participants by mass spectrometry, and the relationship between sialylation and BP was evaluated. Effects of normalizing IgG sialylation were determined in HFD-fed mice administered the sialic acid precursor N-acetyl-D-mannosamine (ManNAc). RESULTS: Mice deficient in B cells were protected from obesity-induced hypertension. Compared with IgG from control chow-fed mice, IgG from HFD-fed mice was hyposialylated, and it raised BP when transferred to recipients lacking IgG; the hypertensive response was absent if recipients were FcγRIIB-deficient. Neuraminidase-treated IgG lacking the Fc glycan terminal sialic acid also raised BP. In cultured endothelial cells, via FcγRIIB, IgG from HFD-fed mice and neuraminidase-treated IgG inhibited vascular endothelial growth factor activation of endothelial NO synthase by altering endothelial NO synthase phosphorylation. In humans, obesity was associated with lower IgG sialylation, and systolic BP was inversely related to IgG sialylation. Mice deficient in FcγRIIB in endothelium were protected from obesity-induced hypertension. Furthermore, in HFD-fed mice, ManNAc normalized IgG sialylation and prevented obesity-induced hypertension. CONCLUSIONS: Hyposialylated IgG and FcγRIIB in endothelium are critically involved in obesity-induced hypertension in mice, and supportive evidence was obtained in humans. Interventions targeting these mechanisms, such as ManNAc supplementation, may provide novel means to break the link between obesity and hypertension.

SR-B1 drives endothelial cell LDL transcytosis via DOCK4 to promote atherosclerosis.

Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke1, is initiated by passage of low-density lipoprotein (LDL) cholesterol into the artery wall and its engulfment by macrophages, which leads to foam cell formation and lesion development2,3. It is unclear how circulating LDL enters the artery wall to instigate atherosclerosis. Here we show in mice that scavenger receptor class B type 1 (SR-B1) in endothelial cells mediates the delivery of LDL into arteries and its accumulation by artery wall macrophages, thereby promoting atherosclerosis. LDL particles are colocalized with SR-B1 in endothelial cell intracellular vesicles in vivo, and transcytosis of LDL across endothelial monolayers requires its direct binding to SR-B1 and an eight-amino-acid cytoplasmic domain of the receptor that recruits the guanine nucleotide exchange factor dedicator of cytokinesis 4 (DOCK4)4. DOCK4 promotes internalization of SR-B1 and transport of LDL by coupling the binding of LDL to SR-B1 with activation of RAC1. The expression of SR-B1 and DOCK4 is increased in atherosclerosis-prone regions of the mouse aorta before lesion formation, and in human atherosclerotic arteries when compared with normal arteries. These findings challenge the long-held concept that atherogenesis involves passive movement of LDL across a compromised endothelial barrier. Interventions that inhibit the endothelial delivery of LDL into artery walls may represent a new therapeutic category in the battle against cardiovascular disease.

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance.

Type 2 diabetes mellitus (T2DM) is a common complication of obesity. Here, we have shown that activation of the IgG receptor FcγRIIB in endothelium by hyposialylated IgG plays an important role in obesity-induced insulin resistance. Despite becoming obese on a high-fat diet (HFD), mice lacking FcγRIIB globally or selectively in endothelium were protected from insulin resistance as a result of the preservation of insulin delivery to skeletal muscle and resulting maintenance of muscle glucose disposal. IgG transfer in IgG-deficient mice implicated IgG as the pathogenetic ligand for endothelial FcγRIIB in obesity-induced insulin resistance. Moreover, IgG transferred from patients with T2DM but not from metabolically healthy subjects caused insulin resistance in IgG-deficient mice via FcγRIIB, indicating that similar processes may be operative in T2DM in humans. Mechanistically, the activation of FcγRIIB by IgG from obese mice impaired endothelial cell insulin transcytosis in culture and in vivo. These effects were attributed to hyposialylation of the Fc glycan, and IgG from T2DM patients was also hyposialylated. In HFD-fed mice, supplementation with the sialic acid precursor N-acetyl-D-mannosamine restored IgG sialylation and preserved insulin sensitivity without affecting weight gain. Thus, IgG sialylation and endothelial FcγRIIB may represent promising therapeutic targets to sever the link between obesity and T2DM.

Endothelial Fcγ Receptor IIB Activation Blunts Insulin Delivery to Skeletal Muscle to Cause Insulin Resistance in Mice.

Modest elevations in C-reactive protein (CRP) are associated with type 2 diabetes. We previously revealed in mice that increased CRP causes insulin resistance and mice globally deficient in the CRP receptor Fcγ receptor IIB (FcγRIIB) were protected from the disorder. FcγRIIB is expressed in numerous cell types including endothelium and B lymphocytes. Here we investigated how endothelial FcγRIIB influences glucose homeostasis, using mice with elevated CRP expressing or lacking endothelial FcγRIIB. Whereas increased CRP caused insulin resistance in mice expressing endothelial FcγRIIB, mice deficient in the endothelial receptor were protected. The insulin resistance with endothelial FcγRIIB activation was due to impaired skeletal muscle glucose uptake caused by attenuated insulin delivery, and it was associated with blunted endothelial nitric oxide synthase (eNOS) activation in skeletal muscle. In culture, CRP suppressed endothelial cell insulin transcytosis via FcγRIIB activation and eNOS antagonism. Furthermore, in knock-in mice harboring constitutively active eNOS, elevated CRP did not invoke insulin resistance. Collectively these findings reveal that by inhibiting eNOS, endothelial FcγRIIB activation by CRP blunts insulin delivery to skeletal muscle to cause insulin resistance. Thus, a series of mechanisms in endothelium that impairs insulin movement has been identified that may contribute to type 2 diabetes pathogenesis.

Loss of Reelin protects against atherosclerosis by reducing leukocyte-endothelial cell adhesion and lesion macrophage accumulation.

The multimodular glycoprotein Reelin controls neuronal migration and synaptic transmission by binding to apolipoprotein E receptor 2 (Apoer2) and very low density lipoprotein receptor (Vldlr) on neurons. In the periphery, Reelin is produced by the liver, circulates in blood, and promotes thrombosis and hemostasis. To investigate if Reelin influences atherogenesis, we studied atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice in which we inducibly deleted Reelin either ubiquitously or only in the liver, thus preventing the production of circulating Reelin. In both types of Reelin-deficient mice, atherosclerosis progression was markedly attenuated, and macrophage content and endothelial cell staining for vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were reduced at the sites of atherosclerotic lesions. Intravital microscopy revealed decreased leukocyte-endothelial adhesion in the Reelin-deficient mice. In cultured human endothelial cells, Reelin enhanced monocyte adhesion and increased ICAM1, VCAM1, and E-selectin expression by suppressing endothelial nitric oxide synthase (eNOS) activity and increasing nuclear factor κB (NF-κB) activity in an Apoer2-dependent manner. These findings suggest that circulating Reelin promotes atherosclerosis by increasing vascular inflammation, and that reducing or inhibiting circulating Reelin may present a novel approach for the prevention of cardiovascular disease.

IgG receptor FcγRIIB plays a key role in obesity-induced hypertension.

There is a well-recognized association between obesity, inflammation, and hypertension. Why obesity causes hypertension is poorly understood. We previously demonstrated using a C-reactive protein (CRP) transgenic mouse that CRP induces hypertension that is related to NO deficiency. Our prior work in cultured endothelial cells identified the Fcγ receptor IIB (FcγRIIB) as the receptor for CRP whereby it antagonizes endothelial NO synthase. Recognizing known associations between CRP and obesity and hypertension in humans, in the present study we tested the hypothesis that FcγRIIB plays a role in obesity-induced hypertension in mice. Using radiotelemetry, we first demonstrated that the hypertension observed in transgenic mouse-CRP is mediated by the receptor, indicating that FcγRIIB is capable of modifying blood pressure. We then discovered in a model of diet-induced obesity yielding equal adiposity in all study groups that whereas FcγRIIB(+/+) mice developed obesity-induced hypertension, FcγRIIB(-/-) mice were fully protected. Levels of CRP, the related pentraxin serum amyloid P component which is the CRP-equivalent in mice, and total IgG were unaltered by diet-induced obesity; FcγRIIB expression in endothelium was also unchanged. However, whereas IgG isolated from chow-fed mice had no effect, IgG from high-fat diet-fed mice inhibited endothelial NO synthase in cultured endothelial cells, and this was an FcγRIIB-dependent process. Thus, we have identified a novel role for FcγRIIB in the pathogenesis of obesity-induced hypertension, independent of processes regulating adiposity, and it may entail an IgG-induced attenuation of endothelial NO synthase function. Approaches targeting FcγRIIB may potentially offer new means to treat hypertension in obese individuals.

HDL cholesterol efflux capacity and incident cardiovascular events.

BACKGROUND: It is unclear whether high-density lipoprotein (HDL) cholesterol concentration plays a causal role in atherosclerosis. A more important factor may be HDL cholesterol efflux capacity, the ability of HDL to accept cholesterol from macrophages, which is a key step in reverse cholesterol transport. We investigated the epidemiology of cholesterol efflux capacity and its association with incident atherosclerotic cardiovascular disease outcomes in a large, multiethnic population cohort. METHODS: We measured HDL cholesterol level, HDL particle concentration, and cholesterol efflux capacity at baseline in 2924 adults free from cardiovascular disease who were participants in the Dallas Heart Study, a probability-based population sample. The primary end point was atherosclerotic cardiovascular disease, defined as a first nonfatal myocardial infarction, nonfatal stroke, or coronary revascularization or death from cardiovascular causes. The median follow-up period was 9.4 years. RESULTS: In contrast to HDL cholesterol level, which was associated with multiple traditional risk factors and metabolic variables, cholesterol efflux capacity had minimal association with these factors. Baseline HDL cholesterol level was not associated with cardiovascular events in an adjusted analysis (hazard ratio, 1.08; 95% confidence interval [CI], 0.59 to 1.99). In a fully adjusted model that included traditional risk factors, HDL cholesterol level, and HDL particle concentration, there was a 67% reduction in cardiovascular risk in the highest quartile of cholesterol efflux capacity versus the lowest quartile (hazard ratio, 0.33; 95% CI, 0.19 to 0.55). Adding cholesterol efflux capacity to traditional risk factors was associated with improvement in discrimination and reclassification indexes. CONCLUSIONS: Cholesterol efflux capacity, a new biomarker that characterizes a key step in reverse cholesterol transport, was inversely associated with the incidence of cardiovascular events in a population-based cohort. (Funded by the Donald W. Reynolds Foundation and others.).

Genetic variants of ApoE and ApoER2 differentially modulate endothelial function.

It is poorly understood why there is greater cardiovascular disease risk associated with the apolipoprotein E4 (apoE) allele vs. apoE3, and also greater risk with the LRP8/apolipoprotein E receptor 2 (ApoER2) variant ApoER2-R952Q. Little is known about the function of the apoE-ApoER2 tandem outside of the central nervous system. We now report that in endothelial cells apoE3 binding to ApoER2 stimulates endothelial NO synthase (eNOS) and endothelial cell migration, and it also attenuates monocyte-endothelial cell adhesion. However, apoE4 does not stimulate eNOS or endothelial cell migration or dampen cell adhesion, and alternatively it selectively antagonizes apoE3/ApoER2 actions. The contrasting endothelial actions of apoE4 vs. apoE3 require the N-terminal to C-terminal interaction in apoE4 that distinguishes it structurally from apoE3. Reconstitution experiments further reveal that ApoER2-R952Q is a loss-of-function variant of the receptor in endothelium. Carotid artery reendothelialization is decreased in ApoER2(-/-) mice, and whereas adenoviral-driven apoE3 expression in wild-type mice has no effect, apoE4 impairs reendothelialization. Moreover, in a model of neointima formation invoked by carotid artery endothelial denudation, ApoER2(-/-) mice display exaggerated neointima development. Thus, the apoE3/ApoER2 tandem promotes endothelial NO production, endothelial repair, and endothelial anti-inflammatory properties, and it prevents neointima formation. In contrast, apoE4 and ApoER2-R952Q display dominant-negative action and loss of function, respectively. Thus, genetic variants of apoE and ApoER2 impact cardiovascular health by differentially modulating endothelial function.

27-Hydroxycholesterol promotes cell-autonomous, ER-positive breast cancer growth.

To date, estrogen is the only known endogenous estrogen receptor (ER) ligand that promotes ER+ breast tumor growth. We report that the cholesterol metabolite 27-hydroxycholesterol (27HC) stimulates MCF-7 cell xenograft growth in mice. More importantly, in ER+ breast cancer patients, 27HC content in normal breast tissue is increased compared to that in cancer-free controls, and tumor 27HC content is further elevated. Increased tumor 27HC is correlated with diminished expression of CYP7B1, the 27HC metabolizing enzyme, and reduced expression of CYP7B1 in tumors is associated with poorer patient survival. Moreover, 27HC is produced by MCF-7 cells, and it stimulates cell-autonomous, ER-dependent, and GDNF-RET-dependent cell proliferation. Thus, 27HC is a locally modulated, nonaromatized ER ligand that promotes ER+ breast tumor growth.

LXRß/estrogen receptor-α signaling in lipid rafts preserves endothelial integrity.

Liver X receptors (LXR) are stimulated by cholesterol-derived oxysterols and serve as transcription factors to regulate gene expression in response to alterations in cholesterol. In the present study, we investigated the role of LXRs in vascular endothelial cells (ECs) and discovered that LXRß has nonnuclear function and stimulates EC migration by activating endothelial NOS (eNOS). This process is mediated by estrogen receptor-α (ERα). LXR activation promoted the direct binding of LXRß to the ligand-binding domain of ERα and initiated an extranuclear signaling cascade that requires ERα Ser118 phosphorylation by PI3K/AKT. Further studies revealed that LXRß and ERα are colocalized and functionally coupled in EC plasma membrane caveolae/lipid rafts. In isolated aortic rings, LXR activation of NOS caused relaxation, while in mice, LXR activation stimulated carotid artery reendothelialization via LXRß- and ERα-dependent processes. These studies demonstrate that LXRß has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.

Point mutations in the ERα Gαi binding domain segregate nonnuclear from nuclear receptor function.

A subpopulation of plasma membrane-associated estrogen receptor (ER)α interact directly with G proteins and mediate nonnuclear receptor signaling. This mechanism underlies numerous processes, including important cardiovascular protective actions of estradiol (E(2)), such as the activation of endothelial NO synthase (eNOS) and endothelial cell growth and migration. In the present work we sought a genetic approach to differentiate nonnuclear from nuclear ERα actions. We generated single alanine substitutions within the Gαi-binding domain of ERα (amino acids 251-260) and tested signaling to eNOS or ERK1,2 and activation of luciferase (Luc) reporters signifying transactivation via direct or indirect ERα-DNA binding in HeLa cells. The point mutants ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A were all incapable of activating eNOS in response to E(2), and ERα-R256A and ERα-D258A also showed loss of ERK1,2 activation. In contrast, ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A all displayed normal capacity to invoke E(2)-induced transactivation of estrogen response element (ERE)-Luc or Sp1-Luc. However, whereas activator protein 1-Luc activation by ERα-R256A and ERα-D258A was intact, ERα-K257A and ERα-R260A were incapable of activator protein 1-Luc activation. In in vitro pull-down assays with the two mutants that lack all nonnuclear functions tested and retain all nuclear functions tested, ERα-R256A and ERα-D258A, there was normal direct interaction between Gαi and ERα-R256A and an absence of interaction between Gαi and ERα-D258A. When expressed in endothelial cells, these two mutants prevented E(2)-induced migration and eNOS activation mediated by endogenous receptor, indicative of dominant-negative action. Thus, the point mutants ERα-R256A and ERα-D258A in the receptor GαI-binding domain provide genetic segregation of nonnuclear from nuclear ERα function.

Scavenger receptor class B type I is a plasma membrane cholesterol sensor.

RATIONALE: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. OBJECTIVE: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. METHODS AND RESULTS: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-ß-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-ß-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. CONCLUSIONS: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.

Coupling of Fcγ receptor I to Fcγ receptor IIb by SRC kinase mediates C-reactive protein impairment of endothelial function.

RATIONALE: Elevations in C-reactive protein (CRP) are associated with increased cardiovascular disease risk and endothelial dysfunction. CRP antagonizes endothelial nitric oxide synthase (eNOS) through processes mediated by the IgG receptor Fcγ receptor IIB (FcγRIIB), its immunoreceptor tyrosine-based inhibitory motif, and SH2 domain-containing inositol 5'-phosphatase 1. In mice, CRP actions on eNOS blunt carotid artery re-endothelialization. OBJECTIVE: How CRP activates FcγRIIB in endothelium is not known. We determined the role of Fcγ receptor I (FcγRI) and the basis for coupling of FcγRI to FcγRIIB in endothelium. METHODS AND RESULTS: In cultured endothelial cells, FcγRI-blocking antibodies prevented CRP antagonism of eNOS, and CRP activated Src via FcγRI. CRP-induced increases in FcγRIIB immunoreceptor tyrosine-based inhibitory motif phosphorylation and SH2 domain-containing inositol 5'-phosphatase 1 activation were Src-dependent, and Src inhibition prevented eNOS antagonism by CRP. Similar processes mediated eNOS antagonism by aggregated IgG used to mimic immune complex. Carotid artery re-endothelialization was evaluated in offspring from crosses of CRP transgenic mice (TG-CRP) with either mice lacking the γ subunit of FcγRI (FcRγ(-/-)) or FcγRIIB(-/-) mice. Whereas re-endothelialization was impaired in TG-CRP vs wild-type, it was normal in both FcRγ(-/-); TG-CRP and FcγRIIB(-/-); TG-CRP mice. CONCLUSIONS: CRP antagonism of eNOS is mediated by the coupling of FcγRI to FcγRIIB by Src kinase and resulting activation of SH2 domain-containing inositol 5'-phosphatase 1, and consistent with this mechanism, both FcγRI and FcγRIIB are required for CRP to blunt endothelial repair in vivo. Similar mechanisms underlie eNOS antagonism by immune complex. FcγRI and FcγRIIB may be novel therapeutic targets for preventing endothelial dysfunction in inflammatory or immune complex-mediated conditions.

Antiphospholipid antibodies promote leukocyte-endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via ß2GPI and apoER2.

In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to ß2 glycoprotein I (ß2GPI) induce endothelial cell-leukocyte adhesion and thrombus formation via unknown mechanisms. Here we show that in mice both of these processes are caused by the inhibition of eNOS. In studies of cultured human, bovine, and mouse endothelial cells, the promotion of monocyte adhesion by aPL entailed decreased bioavailable NO, and aPL fully antagonized eNOS activation by diverse agonists. Similarly, NO-dependent, acetylcholine-induced increases in carotid vascular conductance were impaired in aPL-treated mice. The inhibition of eNOS was caused by antibody recognition of domain I of ß2GPI and ß2GPI dimerization, and it was due to attenuated eNOS S1179 phosphorylation mediated by protein phosphatase 2A (PP2A). Furthermore, LDL receptor family member antagonism with receptor-associated protein (RAP) prevented aPL inhibition of eNOS in cell culture, and ApoER2-/- mice were protected from aPL inhibition of eNOS in vivo. Moreover, both aPL-induced increases in leukocyte-endothelial cell adhesion and thrombus formation were absent in eNOS-/- and in ApoER2-/- mice. Thus, aPL-induced leukocyte-endothelial cell adhesion and thrombosis are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by ß2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.

Non-nuclear estrogen receptor alpha signaling promotes cardiovascular protection but not uterine or breast cancer growth in mice.

Steroid hormone receptors function classically in the nucleus as transcription factors. However, recent data indicate that there are also non-nuclear subpopulations of steroid hormone receptors, including estrogen receptors (ERs), that mediate membrane-initiated signaling of unclear basis and significance. Here we have shown that an estrogen-dendrimer conjugate (EDC) that is excluded from the nucleus stimulates endothelial cell proliferation and migration via ERalpha, direct ERalpha-Galphai interaction, and endothelial NOS (eNOS) activation. Analysis of mice carrying an estrogen response element luciferase reporter, ER-regulated genes in the mouse uterus, and eNOS enzyme activation further indicated that EDC specifically targets non-nuclear processes in vivo. In mice, estradiol and EDC equally stimulated carotid artery reendothelialization in an ERalpha- and G protein-dependent manner, and both agents attenuated the development of neointimal hyperplasia following endothelial injury. In contrast, endometrial carcinoma cell growth in vitro and uterine enlargement and MCF-7 cell breast cancer xenograft growth in vivo were stimulated by estradiol but not EDC. Thus, EDC is a non-nuclear selective ER modulator (SERM) in vivo, and in mice, non-nuclear ER signaling promotes cardiovascular protection. These processes potentially could be harnessed to provide vascular benefit without increasing the risk of uterine or breast cancer.

C-reactive protein inhibits insulin activation of endothelial nitric oxide synthase via the immunoreceptor tyrosine-based inhibition motif of FcgammaRIIB and SHIP-1.

Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor FcgammaRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcgammaRIIB in NIH3T3IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domain-containing inositol 5'-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via FcgammaRIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes.

Postnatal estradiol up-regulates lung nitric oxide synthases and improves lung function in bronchopulmonary dysplasia.

RATIONALE: Nitric oxide (NO) plays an important role in lung development and perinatal lung function, and pulmonary NO synthases (NOS) are decreased in bronchopulmonary dysplasia (BPD) following preterm birth. Fetal estradiol levels increase during late gestation and estradiol up-regulates NOS, suggesting that after preterm birth estradiol deprivation causes attenuated lung NOS resulting in impaired pulmonary function. OBJECTIVE: To test the effects of postnatal estradiol administration in a primate model of BPD over 14 days after delivery at 125 days of gestation (term = 185 d). METHODS: Cardiopulmonary function was assessed by echocardiography and whole body plethysmography. Lung morphometric and histopathologic analyses were performed, and NOS enzymatic activity and abundance were measured. MEASUREMENTS AND MAIN RESULTS: Estradiol caused an increase in blood pressure and ductus arteriosus closure. Expiratory resistance and lung compliance were also improved, and this occurred before spontaneous ductal closure. Furthermore, both oxygenation and ventilation indices were improved with estradiol, and the changes in lung function and ventilatory support requirements persisted throughout the study period. Whereas estradiol had negligible effect on indicators of lung inflammation and on lung structure assessed after the initial 14 days of ventilatory support, it caused an increase in lung neuronal and endothelial NOS enzymatic activity. CONCLUSIONS: In a primate model of BPD, postnatal estradiol treatment had favorable cardiovascular impact, enhanced pulmonary function, and lowered requirements for ventilatory support in association with an up-regulation of lung NOS. Estradiol may be an efficacious postnatal therapy to improve lung function and outcome in preterm infants.

The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity.

Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1-/- mice, and this phenotype persists in PDZK1-/- mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity.

27-Hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen.

The cardioprotective effects of estrogen are mediated by receptors expressed in vascular cells. Here we show that 27-hydroxycholesterol (27HC), an abundant cholesterol metabolite that is elevated with hypercholesterolemia and found in atherosclerotic lesions, is a competitive antagonist of estrogen receptor action in the vasculature. 27HC inhibited both the transcription-mediated and the non-transcription-mediated estrogen-dependent production of nitric oxide by vascular cells, resulting in reduced estrogen-induced vasorelaxation of rat aorta. Furthermore, increasing 27HC levels in mice by diet-induced hypercholesterolemia, pharmacologic administration or genetic manipulation (by knocking out the gene encoding the catabolic enzyme CYP7B1) decreased estrogen-dependent expression of vascular nitric oxide synthase and repressed carotid artery reendothelialization. As well as antiestrogenic effects, there were proestrogenic actions of 27HC that were cell-type specific, indicating that 27HC functions as an endogenous selective estrogen receptor modulator (SERM). Taken together, these studies point to 27HC as a contributing factor in the loss of estrogen protection from vascular disease.

Nitric oxide attenuates epithelial-mesenchymal transition in alveolar epithelial cells.

Patients with interstitial lung diseases, such as idiopathic pulmonary fibrosis (IPF) and bronchopulmonary dysplasia (BPD), suffer from lung fibrosis secondary to myofibroblast-mediated excessive ECM deposition and destruction of lung architecture. Transforming growth factor (TGF)-beta1 induces epithelial-mesenchymal transition (EMT) of alveolar epithelial cells (AEC) to myofibroblasts both in vitro and in vivo. Inhaled nitric oxide (NO) attenuates ECM accumulation, enhances lung growth, and decreases alveolar myofibroblast number in experimental models. We therefore hypothesized that NO attenuates TGF-beta1-induced EMT in cultured AEC. Studies of the capacity for endogenous NO production in AEC revealed that endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) are expressed and active in AEC. Total NOS activity was 1.3 pmol x mg protein(-1) x min(-1) with 67% derived from eNOS. TGF-beta1 (50 pM) suppressed eNOS expression by more than 60% and activity by 83% but did not affect iNOS expression or activity. Inhibition of endogenous NOS with l-NAME led to spontaneous EMT, manifested by increased alpha-smooth muscle actin (alpha-SMA) expression and a fibroblast-like morphology. Provision of exogenous NO to TGF-beta1-treated AEC decreased stress fiber-associated alpha-SMA expression and decreased collagen I expression by 80%. NO-treated AEC also retained an epithelial morphology and expressed increased lamellar protein, E-cadherin, and pro-surfactant protein B compared with those treated with TGF-beta alone. These findings indicate that NO serves a critical role in preserving an epithelial phenotype and in attenuating EMT in AEC. NO-mediated regulation of AEC fate may have important implications in the pathophysiology and treatment of diseases such as IPF and BPD.