Protein Phosphatase 2A Activation Via ApoER2 in Trophoblasts Drives Preeclampsia in a Mouse Model of the Antiphospholipid Syndrome.

[Figure: see text].

A Protein Kinase C Phosphorylation Motif in GLUT1 Affects Glucose Transport and is Mutated in GLUT1 Deficiency Syndrome.

Protein kinase C has been implicated in the phosphorylation of the erythrocyte/brain glucose transporter, GLUT1, without a clear understanding of the site(s) of phosphorylation and the possible effects on glucose transport. Through in vitro kinase assays, mass spectrometry, and phosphospecific antibodies, we identify serine 226 in GLUT1 as a PKC phosphorylation site. Phosphorylation of S226 is required for the rapid increase in glucose uptake and enhanced cell surface localization of GLUT1 induced by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Endogenous GLUT1 is phosphorylated on S226 in primary endothelial cells in response to TPA or VEGF. Several naturally occurring, pathogenic mutations that cause GLUT1 deficiency syndrome disrupt this PKC phosphomotif, impair the phosphorylation of S226 in vitro, and block TPA-mediated increases in glucose uptake. We demonstrate that the phosphorylation of GLUT1 on S226 regulates glucose transport and propose that this modification is important in the physiological regulation of glucose transport.

Reelin Depletion Protects Against Atherosclerosis by Decreasing Vascular Adhesion of Leukocytes.

[Figure: see text].

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.

Antiphospholipid antibodies induce thrombosis by PP2A activation via apoER2-Dab2-SHC1 complex formation in endothelium.

In the antiphospholipid syndrome (APS), antiphospholipid antibody (aPL) recognition of ß2 glycoprotein I promotes thrombosis, and preclinical studies indicate that this is due to endothelial nitric oxide synthase (eNOS) antagonism via apolipoprotein E receptor 2 (apoER2)-dependent processes. How apoER2 molecularly links these events is unknown. Here, we show that, in endothelial cells, the apoER2 cytoplasmic tail serves as a scaffold for aPL-induced assembly and activation of the heterotrimeric protein phosphatase 2A (PP2A). Disabled-2 (Dab2) recruitment to the apoER2 NPXY motif promotes the activating L309 methylation of the PP2A catalytic subunit by leucine methyl transferase-1. Concurrently, Src homology domain-containing transforming protein 1 (SHC1) recruits the PP2A scaffolding subunit to the proline-rich apoER2 C terminus along with 2 distinct regulatory PP2A subunits that mediate inhibitory dephosphorylation of Akt and eNOS. In mice, the coupling of these processes in endothelium is demonstrated to underlie aPL-invoked thrombosis. By elucidating these intricacies in the pathogenesis of APS-related thrombosis, numerous potential new therapeutic targets have been identified.

New Insights in the Pathophysiology of Antiphospholipid Syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disorder characterized by an elevated risk for arterial and venous thrombosis and pregnancy-related morbidity. Since the discovery of the disease in 1980s, numerous studies in cell culture systems, in animal models, and in patient populations have been reported, leading to a deeper understanding of the pathogenesis of APS. These studies have determined that circulating autoantibodies, collectively called antiphospholipid antibodies (aPL), the majority of which recognize cell surface proteins attached to the plasma membrane phospholipids, play a causal role in the development of the disease. The binding of aPL to the cell surface antigens triggers interaction of the complex with transmembrane receptors to initiate intracellular signaling in critical cell types, including platelets, monocytes, endothelial cells, and trophoblasts. Subsequent alteration of various cell functions results in inflammation, thrombus formation, and pregnancy complications. Apolipoprotein E receptor 2 (apoER2), a lipoprotein receptor family member, has been implicated as a mediator for aPL actions in platelets and endothelial cells. Nitric oxide (NO) is a signaling molecule known to exert potent antithrombotic, anti-inflammatory, and anti-atherogenic effects. NO insufficiency and oxidative stress have been linked to APS pathogenesis. This review will focus on the recent findings on how apoER2 and dysregulation of NO production contribute to aPL-mediated pathologies in APS.

ETS-related gene (ERG) controls endothelial cell permeability via transcriptional regulation of the claudin 5 (CLDN5) gene.

ETS-related gene (ERG) is a member of the ETS transcription factor family. Our previous studies have shown that ERG expression is highly enriched in endothelial cells (EC) both in vitro and in vivo. ERG expression is markedly repressed in response to inflammatory stimuli. It has been shown that ERG is a positive regulator of several EC-restricted genes including VE-cadherin, endoglin, and von Willebrand factor, and a negative regulator of other genes such as interleukin (IL)-8 and intercellular adhesion molecule (ICAM)-1. In this study we have identified a novel role for ERG in the regulation of EC barrier function. ERG knockdown results in marked increases in EC permeability. This is associated with a significant increase of stress fiber and gap formation in EC. Furthermore, we identify CLDN5 as a downstream target of ERG in EC. Thus, our results suggest that ERG plays a pivotal role in regulating EC barrier function and that this effect is mediated in part through its regulation of CLDN5 gene expression.

RhoJ is an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG.

ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knock-down on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knock-down of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver.

Endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle.

The estrogen receptor (ER) designated ERα has actions in many cell and tissue types that impact glucose homeostasis. It is unknown if these include mechanisms in endothelial cells, which have the potential to influence relative obesity, and processes in adipose tissue and skeletal muscle that impact glucose control. Here we show that independent of impact on events in adipose tissue, endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle. Endothelial ERα-deficient male mice are glucose intolerant and insulin resistant, and in females the antidiabetogenic actions of estradiol (E2) are absent. The glucose dysregulation is due to impaired skeletal muscle glucose disposal that results from attenuated muscle insulin delivery. Endothelial ERα activation stimulates insulin transcytosis by skeletal muscle microvascular endothelial cells. Mechanistically this involves nuclear ERα-dependent upregulation of vesicular trafficking regulator sorting nexin 5 (SNX5) expression, and PI3 kinase activation that drives plasma membrane recruitment of SNX5. Thus, coupled nuclear and non-nuclear actions of ERα promote endothelial insulin transport to skeletal muscle to foster normal glucose homeostasis.

Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice.

Hypercholesterolemia and vascular inflammation are key interconnected contributors to the pathogenesis of atherosclerosis. How hypercholesterolemia initiates vascular inflammation is poorly understood. Here we show in male mice that hypercholesterolemia-driven endothelial activation, monocyte recruitment and atherosclerotic lesion formation are promoted by a crosstalk between macrophages and endothelial cells mediated by the cholesterol metabolite 27-hydroxycholesterol (27HC). The pro-atherogenic actions of macrophage-derived 27HC require endothelial estrogen receptor alpha (ERα) and disassociation of the cytoplasmic scaffolding protein septin 11 from ERα, leading to extranuclear ERα- and septin 11-dependent activation of NF-κB. Furthermore, pharmacologic inhibition of cyp27a1, which generates 27HC, affords atheroprotection by reducing endothelial activation and monocyte recruitment. These findings demonstrate cell-to-cell communication by 27HC, and identify a major causal linkage between the hypercholesterolemia and vascular inflammation that partner to promote atherosclerosis. Interventions interrupting this linkage may provide the means to blunt vascular inflammation without impairing host defense to combat the risk of atherosclerotic cardiovascular disease that remains despite lipid-lowering therapies.

Endothelial lumen signaling complexes control 3D matrix-specific tubulogenesis through interdependent Cdc42- and MT1-MMP-mediated events.

Here, we define an endothelial cell (EC) lumen signaling complex involving Cdc42, Par6b, Par3, junction adhesion molecule (Jam)-B and Jam-C, membrane type 1-matrix metalloproteinase (MT1-MMP), and integrin alpha(2)beta(1), which coassociate to control human EC tubulogenesis in 3D collagen matrices. Blockade of both Jam-B and Jam-C using antibodies, siRNA, or dominant-negative mutants completely interferes with lumen and tube formation resulting from a lack of Cdc42 activation, inhibition of Cdc42-GTP-dependent signal transduction, and blockade of MT1-MMP-dependent proteolysis. This process requires interdependent Cdc42 and MT1-MMP signaling, which involves Par3 binding to the Jam-B and Jam-C cytoplasmic tails, an interaction that is necessary to physically couple the components of the lumen signaling complex. MT1-MMP proteolytic activity is necessary for Cdc42 activation during EC tube formation in 3D collagen matrices but not on 2D collagen surfaces, whereas Cdc42 activation is necessary for MT1-MMP to create vascular guidance tunnels and tube networks in 3D matrices through proteolytic events. This work reveals a novel interdependent role for Cdc42-dependent signaling and MT1-MMP-dependent proteolysis, a process that occurs selectively in 3D collagen matrices and that requires EC lumen signaling complexes, to control human EC tubulogenesis during vascular morphogenesis.

Molecular mechanisms controlling vascular lumen formation in three-dimensional extracellular matrices.

Considerable progress has been made toward a molecular understanding of how cells form lumen and tube structures in three-dimensional (3D) extracellular matrices (ECM). This progress has occurred through work performed with endothelial and epithelial cell models using both in vitro and in vivo approaches. Despite the apparent similarities between endothelial and epithelial cell lumen and tube formation mechanisms, there are clear distinctions that directly relate to their functional differences. This review will focus on endothelial cell (EC) lumen formation mechanisms which control blood vessel formation during development and postnatal life. Of great interest is that an EC lumen signaling complex has been identified which controls human EC lumen and tube formation in 3D matrices and which coordinates integrin-ECM contacts, cell surface proteolysis, cytoskeletal rearrangements, and cell polarity. This complex consists of the collagen-binding integrin α2ß1, the collagen-degrading membrane-type 1 matrix metalloproteinase (MT1-MMP), junction adhesion molecule (Jam)C, JamB, polarity proteins Par3 and Par6b, and the Rho GTPase Cdc42-GTP. These interacting proteins are necessary to stimulate 3D matrix-specific signaling events (including activation of protein kinase cascades that regulate the actin and microtubule cytoskeletons) to control the formation of EC lumens and tube networks. Also, EC lumen formation is directly coupled to the generation of vascular guidance tunnels, enzymatically generated ECM conduits that facilitate EC tube remodeling and maturation. Mural cells such as pericytes are recruited along EC tubes within these tunnel spaces to control ECM remodeling events resulting in vascular basement membrane matrix assembly, a key step in tube maturation and stabilization.

MT1-MMP- and Cdc42-dependent signaling co-regulate cell invasion and tunnel formation in 3D collagen matrices.

Complex signaling events control tumor invasion in three-dimensional (3D) extracellular matrices. Recent evidence suggests that cells utilize both matrix metalloproteinase (MMP)-dependent and MMP-independent means to traverse 3D matrices. Herein, we demonstrate that lysophosphatidic-acid-induced HT1080 cell invasion requires membrane-type-1 (MT1)-MMP-mediated collagenolysis to generate matrix conduits the width of a cellular nucleus. We define these spaces as single-cell invasion tunnels (SCITs). Once established, cells can migrate within SCITs in an MMP-independent manner. Endothelial cells, smooth muscle cells and fibroblasts also generate SCITs during invasive events, suggesting that SCIT formation represents a fundamental mechanism of cellular motility within 3D matrices. Coordinated cellular signaling events are required during SCIT formation. MT1-MMP, Cdc42 and its associated downstream effectors such as MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) and Pak4 (p21 protein-activated kinase 4), protein kinase Calpha and the Rho-associated coiled-coil-containing protein kinases (ROCK-1 and ROCK-2) coordinate signaling necessary for SCIT formation. Finally, we show that MT1-MMP and Cdc42 are fundamental components of a co-associated invasion-signaling complex that controls directed single-cell invasion of 3D collagen matrices.

Endothelial cell lumen and vascular guidance tunnel formation requires MT1-MMP-dependent proteolysis in 3-dimensional collagen matrices.

Here we show that endothelial cells (EC) require matrix type 1-metalloproteinase (MT1-MMP) for the formation of lumens and tube networks in 3-dimensional (3D) collagen matrices. A fundamental consequence of EC lumen formation is the generation of vascular guidance tunnels within collagen matrices through an MT1-MMP-dependent proteolytic process. Vascular guidance tunnels represent a conduit for EC motility within these spaces (a newly remodeled 2D matrix surface) to both assemble and remodel tube structures. Interestingly, it appears that twice as many tunnel spaces are created than are occupied by tube networks after several days of culture. After tunnel formation, these spaces represent a 2D migratory surface within 3D collagen matrices allowing for EC migration in an MMP-independent fashion. Blockade of EC lumenogenesis using inhibitors that interfere with the process (eg, integrin, MMP, PKC, Src) completely abrogates the formation of vascular guidance tunnels. Thus, the MT1-MMP-dependent proteolytic process that creates tunnel spaces is directly and functionally coupled to the signaling mechanisms required for EC lumen and tube network formation. In summary, a fundamental and previously unrecognized purpose of EC tube morphogenesis is to create networks of matrix conduits that are necessary for EC migration and tube remodeling events critical to blood vessel assembly.

Apolipoprotein E receptor 2 deficiency decreases endothelial adhesion of monocytes and protects against autoimmune encephalomyelitis.

Under normal conditions, the blood-brain barrier effectively regulates the passage of immune cells into the central nervous system (CNS). However, under pathological conditions such as multiple sclerosis (MS), leukocytes, especially monocytes, infiltrate the CNS where they promote inflammatory demyelination, resulting in paralysis. Therapies targeting the immune cells directly and preventing leukocyte infiltration exist for MS but may compromise the immune system. Here, we explore how apolipoprotein E receptor 2 (ApoER2) regulates vascular adhesion and infiltration of monocytes during inflammation. We induced experimental autoimmune encephalitis in ApoER2 knockout mice and in mice carrying a loss-of-function mutation in the ApoER2 cytoplasmic domain. In both models, paralysis and neuroinflammation were largely abolished as a result of greatly diminished monocyte adherence due to reduced expression of adhesion molecules on the endothelial surface. Our findings expand our mechanistic understanding of the vascular barrier, the regulation of inflammation and vascular permeability, and the therapeutic potential of ApoER2-targeted therapies.

Reelin depletion protects against autoimmune encephalomyelitis by decreasing vascular adhesion of leukocytes.

Neuroinflammation as a result of immune cell recruitment into the central nervous system (CNS) is a key pathogenic mechanism of multiple sclerosis (MS). However, current anti-inflammatory interventions depleting immune cells or directly targeting their trafficking into the CNS can have serious side effects, highlighting a need for better immunomodulatory strategies. We detected increased Reelin concentrations in the serum of patients with MS, resulting in increased endothelial permeability to leukocytes through increased nuclear factor κB-mediated expression of vascular adhesion molecules. We thus investigated the prophylactic and therapeutic potential of Reelin immunodepletion in experimental autoimmune encephalomyelitis (EAE) and further validated the results in Reelin knockout mice. Removal of plasma Reelin by either approach protected against neuroinflammation and largely abolished the neurological consequences by reducing endothelial permeability and immune cell accumulation in the CNS. Our findings suggest Reelin depletion as a therapeutic approach with an inherent good safety margin for the treatment of MS and other diseases where leukocyte extravasation is a major driver of pathogenicity.

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.

Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes.

Trypanosome RNA editing by uridylate insertion or deletion cycles is a mitochondrial mRNA maturation process catalyzed by multisubunit complexes. A full-round of editing entails three consecutive steps directed by partially complementary guide RNAs: pre-mRNA cleavage, U addition or removal, and ligation. The structural and functional composition of editing complexes is intensively studied, but their molecular interactions in and around editing sites are not completely understood. In this study, we performed a systematic analysis of distal RNA requirements for full-round insertion and deletion by purified editosomes. We define minimal substrates for efficient editing of A6 and CYb model transcripts, and established a new substrate, RPS12. Important differences were observed in the composition of substrates for insertion and deletion. Furthermore, we also showed for the first time that natural sites can be artificially converted in both directions: from deletion to insertion or from insertion to deletion. Our site conversions enabled a direct comparison of the two editing kinds at common sites during substrate minimization and demonstrate that all basic determinants directing the editosome to carry out full-round insertion or deletion reside within each editing site. Surprisingly, we were able to engineer a deletion site into CYb, which exclusively undergoes insertion in nature.

Preferential interaction of a 25kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei.

Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a approximately 25kDa polypeptide in mitochondrial extracts that exhibits a preferential "zero-distance" photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [(32)P] at the first editing site. The approximately 25kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5'-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.

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.