Publications by authors named "Ivan S Yuhanna"

29 Publications

  • Page 1 of 1

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

Circulation 2019 12 10;140(24):2005-2018. Epub 2019 Oct 10.

University of Texas Southwestern Medical Center, Dallas (J.P., W.V., S.B., I.S.Y., K.T., A.S., H.C., N.C.S., A.R., K.L.C., C.M., P.W.S.).

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 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027951PMC
December 2019

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

Nature 2019 05 24;569(7757):565-569. Epub 2019 Apr 24.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Atherosclerosis, which underlies life-threatening cardiovascular disorders such as myocardial infarction and stroke, 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 development. 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). 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1140-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6631346PMC
May 2019

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

J Clin Invest 2018 01 27;128(1):309-322. Epub 2017 Nov 27.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI89333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5749535PMC
January 2018

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

Diabetes 2016 07 26;65(7):1996-2005. Epub 2016 Apr 26.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2337/db15-1605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915578PMC
July 2016

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

Sci Signal 2016 Mar 15;9(419):ra29. Epub 2016 Mar 15.

Department of Molecular Genetics, University of Texas (UT) Southwestern Medical Center, Dallas, TX 75390, USA. Center for Translational Neurodegeneration Research, UT Southwestern Medical Center, Dallas, TX 75390, USA. Department of Neuroscience, UT Southwestern Medical Center, Dallas, TX 75390, USA. Department of Neurology and Neurotherapeutics, UT Southwestern Medical Center, Dallas, TX 75390, USA. Center for Neuroscience, Department of Neuroanatomy, Albert-Ludwigs-University, 79104 Freiburg, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5053773PMC
http://dx.doi.org/10.1126/scisignal.aad5578DOI Listing
March 2016

HDL cholesterol efflux capacity and incident cardiovascular events.

N Engl J Med 2014 Dec 18;371(25):2383-93. Epub 2014 Nov 18.

From the Division of Cardiology, Department of Internal Medicine (A.R., A.K., J.D.B., E.G.G., C.R.A., I.J.N., J.A.L.), and the Center for Pulmonary and Vascular Biology, Department of Pediatrics (I.S.Y., P.W.S.), University of Texas Southwestern Medical Center, Dallas; the Department of Internal Medicine, Emory University, Atlanta (K.E.W.); and the Departments of Genetics and Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia (D.R.R.).

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.).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1056/NEJMoa1409065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308988PMC
December 2014

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

Hypertension 2015 Feb 3;65(2):456-62. Epub 2014 Nov 3.

From the Department of Pediatrics, Section of Neonatology, Baylor College of Medicine, Houston, TX (N.C.S., B.-M.D.B.); and Department of Internal Medicine, Division of Cardiology, Hypertension Section (W.V.) and Department of Pediatrics, Center for Pulmonary and Vascular Biology (K.T., I.S.Y., K.L.C., C.M., P.W.S.), University of Texas Southwestern Medical Center, Dallas.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/HYPERTENSIONAHA.114.04670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419357PMC
February 2015

Genetic variants of ApoE and ApoER2 differentially modulate endothelial function.

Proc Natl Acad Sci U S A 2014 Sep 2;111(37):13493-8. Epub 2014 Sep 2.

Center for Pulmonary and Vascular Biology, Department of Pediatrics, and

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1402106111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169937PMC
September 2014

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

Cell Rep 2013 Nov 7;5(3):637-45. Epub 2013 Nov 7.

Department of Pediatrics, Division of Pulmonary and Vascular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2013.10.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3950897PMC
November 2013

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

J Clin Invest 2013 Aug 8;123(8):3488-97. Epub 2013 Jul 8.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063, USA.

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 LXRβ 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 LXRβ 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 LXRβ 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 LXRβ- and ERα-dependent processes. These studies demonstrate that LXRβ has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI66533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3726156PMC
August 2013

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

Mol Endocrinol 2013 Jan 14;27(1):2-11. Epub 2012 Dec 14.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2011-1378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545213PMC
January 2013

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

Circ Res 2013 Jan 28;112(1):140-51. Epub 2012 Sep 28.

Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390, USA.

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-β-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-β-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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.112.280081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564583PMC
January 2013

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

Circ Res 2011 Oct 22;109(10):1132-40. Epub 2011 Sep 22.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.111.254573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215097PMC
October 2011

Antiphospholipid antibodies promote leukocyte-endothelial cell adhesion and thrombosis in mice by antagonizing eNOS via β2GPI and apoER2.

J Clin Invest 2011 Jan 1;121(1):120-31. Epub 2010 Dec 1.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

In antiphospholipid syndrome (APS), antiphospholipid antibodies (aPL) binding to β2 glycoprotein I (β2GPI) 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 β2GPI and β2GPI 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 β2GPI, apoER2, and PP2A. Our results suggest that novel therapies for APS can now be developed targeting these mechanisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI39828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3007129PMC
January 2011

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

J Clin Invest 2010 Jul 23;120(7):2319-30. Epub 2010 Jun 23.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI38291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2898582PMC
July 2010

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

Circ Res 2009 Jun 7;104(11):1275-82. Epub 2009 May 7.

Division of Pulmonary and Vascular Biology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.108.192906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2733870PMC
June 2009

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

Am J Respir Crit Care Med 2009 Mar 16;179(6):492-500. Epub 2009 Jan 16.

Department of Pediatrics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1164/rccm.200805-794OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2654978PMC
March 2009

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

Circ Res 2008 Feb 3;102(4):480-7. Epub 2008 Jan 3.

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

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.107.159079DOI Listing
February 2008

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

Nat Med 2007 Oct 16;13(10):1185-92. Epub 2007 Sep 16.

Department of Pharmacology and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 6001 Forest Park Road, Dallas, Texas 75390-9050, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nm1641DOI Listing
October 2007

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

Am J Physiol Lung Cell Mol Physiol 2007 Jul 11;293(1):L212-21. Epub 2007 May 11.

Division of Neonatal-Perinatal Medicine, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9063, USA.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00475.2006DOI Listing
July 2007

Direct interactions with G α i and G βγ mediate nongenomic signaling by estrogen receptor α .

Mol Endocrinol 2007 Jun 3;21(6):1370-80. Epub 2007 Apr 3.

Department of Pediatrics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9063, USA.

Estrogen induces G protein-dependent nongenomic signaling in a variety of cell types via the activation of a plasma membrane-associated subpopulation of estrogen receptor alpha (ER alpha). Using pull-down experiments with purified recombinant proteins, we now demonstrate that ER alpha binds directly to G alpha i and G betagamma. Mutagenesis and the addition of blocking peptide reveals that this occurs via amino acids 251-260 and 271-595 of ER alpha, respectively. Studies of ER alpha complexed with heterotrimeric G proteins further show that estradiol causes the release of both G alpha i and G betagamma without stimulating GTP binding to G alpha i. Moreover, in COS-7 cells, the disruption of ER alpha-G alpha i interaction by deletion mutagenesis of ER alpha or expression of blocking peptide, as well as G betagamma sequestration with beta-adrenergic receptor kinase C terminus, prevents nongenomic responses to estradiol including src and erk activation. In endothelial cells, the disruption of ER alpha-G alpha i interaction prevents estradiol-induced nitric oxide synthase activation and the resulting attenuation of monocyte adhesion that contributes to estrogen-related cardiovascular protection. Thus, through direct interactions, ER alpha mediates a novel mechanism of G protein activation that provides greater diversity of function of both the steroid hormone receptor and G proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2006-0360DOI Listing
June 2007

FcgammaRIIB mediates C-reactive protein inhibition of endothelial NO synthase.

Circ Res 2005 Nov 3;97(11):1124-31. Epub 2005 Nov 3.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

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 Fcgamma receptors, causes parallel okadaic acid-sensitive loss of eNOS function, FcgammaRIIB expression is demonstrable in endothelium, and heterologous expression studies reveal that CRP antagonism of eNOS requires FcgammaRIIB. In FcgammaRIIB(+/+) mice, CRP blunts acetylcholine-induced increases in carotid artery vascular conductance; in contrast, CRP enhances acetylcholine responses in FcgammaRIIB(-/-) mice. Thus FcgammaRIIB mediates CRP inhibition of eNOS via PP2A, providing a mechanistic link between CRP and endothelial dysfunction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/01.RES.0000194323.77203.feDOI Listing
November 2005

Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling.

J Clin Invest 2005 Apr 24;115(4):969-77. Epub 2005 Mar 24.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

The binding of HDL to scavenger receptor-BI (SR-BI) mediates cholesterol movement. HDL also induces multiple cellular signals, which in endothelium occur through SR-BI and converge to activate eNOS. To determine the molecular basis of a signaling event induced by HDL, we examined the proximal mechanisms in HDL activation of eNOS. In endothelial cells, HDL and methyl-beta-cyclodextrin caused comparable eNOS activation, whereas cholesterol-loaded methyl-beta-cyclodextrin had no effect. Phosphatidylcholine-loaded HDL caused greater stimulation than native HDL, and blocking antibody against SR-BI, which prevents cholesterol efflux, prevented eNOS activation. In a reconstitution model in COS-M6 cells, wild-type SR-BI mediated eNOS activation by both HDL and small unilamellar vesicles (SUVs), whereas the SR-BI mutant AVI, which is incapable of efflux to SUV, transmitted signal by only HDL. In addition, eNOS activation by methyl-beta-cyclodextrin was SR-BI dependent. Studies of mutant and chimeric class B scavenger receptors revealed that the C-terminal cytoplasmic PDZ-interacting domain and the C-terminal transmembrane domains of SR-BI are both necessary for HDL signaling. Furthermore, we demonstrated direct binding of cholesterol to the C-terminal transmembrane domain using a photoactivated derivative of cholesterol. Thus, HDL signaling requires cholesterol binding and efflux and C-terminal domains of SR-BI, and SR-BI serves as a cholesterol sensor on the plasma membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI23858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1069105PMC
April 2005

Dissecting the basis of nongenomic activation of endothelial nitric oxide synthase by estradiol: role of ERalpha domains with known nuclear functions.

Mol Endocrinol 2005 Feb 14;19(2):277-89. Epub 2004 Oct 14.

Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

Estradiol stimulates endothelial nitric oxide synthase (eNOS) via the activation of plasma membrane (PM)-associated estrogen receptor (ER) alpha. The process requires Src and erk signaling and eNOS phosphorylation by phosphoinositide 3-kinase (PI3 kinase)-Akt kinase, with Src and PI3 kinase associating with ERalpha upon ligand activation. To delineate the basis of nongenomic eNOS stimulation, the potential roles of ERalpha domains necessary for classical nuclear function were investigated in COS-7 cells. In cross-linking studies, estradiol-17beta (E2) caused PM-associated ERalpha to form dimers. However, eNOS activation by E2 was unaltered for a dimerization-deficient mutant ERalpha (ERalphaL511R). In contrast, ERalpha mutants lacking the nuclear localization signals (NLS), NLS2,3 (ERalphaDelta250-274) or the DNA binding domain (ERalphaDelta185-251), which targeted normally to PM and caveolae/rafts, were incapable of activating eNOS. The loss of NLS2/NLS3 prevented Src and erk activation, and it altered ligand-induced PI3 kinase-ERalpha interaction and prevented eNOS phosphorylation. Loss of the DNA binding domain did not change E2 activation of Src or erk, but ligand-induced PI3 kinase-ERalpha binding and eNOS phosphorylation did not occur. Thus, dimerization is not required for ERalpha coupling to eNOS; however, NLS2/NLS3 plays a role in Src activation, and the DNA binding region is involved in the dynamic interaction between ERalpha and PI3 kinase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/me.2004-0008DOI Listing
February 2005

Hypoxia-induced left ventricular dysfunction in myoglobin-deficient mice.

Am J Physiol Heart Circ Physiol 2003 Nov 24;285(5):H2132-41. Epub 2003 Jul 24.

Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-8573, USA.

Myoglobin-deficient mice are viable and have preserved cardiac function due to their ability to mount a complex compensatory response involving increased vascularization and the induction of the hypoxia gene program (hypoxia-inducible factor-1alpha, endothelial PAS, heat shock protein27, etc.). To further define and explore functional roles for myoglobin, we challenged age- and gender-matched wild-type and myoglobin-null mice to chronic hypoxia (10% oxygen for 1 day to 3 wk). We observed a 30% reduction in cardiac systolic function in the myoglobin mutant mice exposed to chronic hypoxia with no changes observed in the wild-type control hearts. The cardiac dysfunction observed in the hypoxic myoglobin-null mice was reversible with reexposure to normoxic conditions and could be prevented with treatment of an inhibitor of nitric oxide (NO) synthases. These results support the conclusion that hypoxia-induced cardiac dysfunction in myoglobin-null mice occurs via a NO-mediated mechanism. Utilizing enzymatic assays for NO synthases and immunohistochemical analyses, we observed a marked induction of inducible NO synthase in the hypoxic myoglobin mutant ventricle compared with the wild-type hypoxic control ventricle. These new data establish that myoglobin is an important cytoplasmic cardiac hemoprotein that functions in regulating NO homeostasis within cardiomyocytes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpheart.00147.2003DOI Listing
November 2003

Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease.

Am J Physiol Lung Cell Mol Physiol 2003 May 10;284(5):L749-58. Epub 2003 Jan 10.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas 75390, USA.

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (VeNO(STP)) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups. VeNO(STP) remained stable in the range of 0.5-1.0 nl x kg(-1) x min(-1) from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00334.2002DOI Listing
May 2003

Vasomodulation by skeletal muscle-derived nitric oxide requires alpha-syntrophin-mediated sarcolemmal localization of neuronal Nitric oxide synthase.

Circ Res 2003 Mar 13;92(5):554-60. Epub 2003 Feb 13.

Department of Internal Medicine, Division of Hypertension, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Tex 75390-8586, USA.

Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle where it associates with the dystrophin complex at the sarcolemma by binding to the PDZ domain of alpha-syntrophin. Nitric oxide (NO) produced by skeletal muscle nNOS is proposed to regulate blood flow in exercising muscle by diffusing from the skeletal muscle fibers to the nearby microvessels where it attenuates alpha-adrenergic vasoconstriction. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the vasoregulatory effect of skeletal muscle-derived NO. To test this hypothesis, we performed experiments in alpha-syntrophin null mice and in transgenic mice expressing a mutated alpha-syntrophin lacking the PDZ domain (DeltaPDZ), both of which are characterized by reduced sarcolemmal nNOS. We found that modulation of alpha-adrenergic vasoconstriction was greatly impaired in the contracting muscles of the alpha-syntrophin null mice and transgenic DeltaPDZ mice compared with wild-type mice and transgenic mice expressing full-length alpha-syntrophin. These in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the dystrophin-associated protein alpha-syntrophin and may have implications for the development of potential gene therapy strategies to treat muscular dystrophy or other muscle-related diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/01.RES.0000061570.83105.52DOI Listing
March 2003

High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases.

J Biol Chem 2003 Mar 2;278(11):9142-9. Epub 2003 Jan 2.

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

High density lipoprotein (HDL) activates endothelial nitric-oxide synthase (eNOS), leading to increased production of the antiatherogenic molecule NO. A variety of stimuli regulate eNOS activity through signaling pathways involving Akt kinase and/or mitogen-activated protein (MAP) kinase. In the present study, we investigated the role of kinase cascades in HDL-induced eNOS stimulation in cultured endothelial cells and COS M6 cells transfected with eNOS and the HDL receptor, scavenger receptor B-I. HDL (10-50 microg/ml, 20 min) caused eNOS phosphorylation at Ser-1179, and dominant negative Akt inhibited both HDL-mediated phosphorylation and activation of the enzyme. Phosphoinositide 3-kinase (PI3 kinase) inhibition or dominant negative PI3 kinase also blocked the phosphorylation and activation of eNOS by HDL. Studies with genistein and PP2 showed that the nonreceptor tyrosine kinase, Src, is an upstream stimulator of the PI3 kinase-Akt pathway in this paradigm. In addition, HDL activated MAP kinase through PI3 kinase, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition fully attenuated eNOS stimulation by HDL without affecting Akt or eNOS Ser-1179 phosphorylation. Conversely, dominant negative Akt did not alter HDL-induced MAP kinase activation. These results indicate that HDL stimulates eNOS through common upstream, Src-mediated signaling, which leads to parallel activation of Akt and MAP kinases and their resultant independent modulation of the enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M211394200DOI Listing
March 2003

ERbeta has nongenomic action in caveolae.

Mol Endocrinol 2002 May;16(5):938-46

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

ERalpha and ERbeta serve classically as transcription factors, and ERalpha also mediates nongenomic responses to E2 such as the activation of endothelial nitric oxide synthase (eNOS). In contrast, the nongenomic capacities of endogenous ERbeta are poorly understood. We evaluated eNOS activation by E2 in cultured endothelial cells that express endogenous ERbeta to determine whether the ERbeta isoform has nongenomic action and to reveal the subcellular locale of that function. A subpopulation of ERbeta was localized to the endothelial cell plasma membrane, overexpression of ERbeta enhanced rapid eNOS stimulation by E2, and the response to endogenous ER activation was inhibited by the ERbeta-selective antagonist RR-tetrahydrochrysene (THC). eNOS activation through ERbeta was reconstituted and shown to occur independent of ERalpha in COS-7 cells, and ERbeta protein in COS-7 was directed to the plasma membrane. THC also blunted E2 activation of eNOS in isolated endothelial cell plasma membranes. Furthermore, ERbeta protein was detected and THC attenuated E2 stimulation of eNOS in isolated endothelial cell caveolae, and functional ERbeta-eNOS coupling was recapitulated in caveolae from transfected COS-7 cells. These findings in the ER-eNOS signaling paradigm indicate that endogenous ERbeta has nongenomic action in caveolae.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1210/mend.16.5.0827DOI Listing
May 2002