Publications by authors named "Anastasia Sacharidou"

26 Publications

  • Page 1 of 1

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

Arterioscler Thromb Vasc Biol 2021 Apr 25;41(4):1309-1318. Epub 2021 Feb 25.

Department of Molecular Genetics (L.C., X.X., J.H.), University of Texas (UT) Southwestern Medical Center, Dallas.

[Figure: see text].
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http://dx.doi.org/10.1161/ATVBAHA.121.316000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990715PMC
April 2021

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

Sci Transl Med 2020 08;12(556)

Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

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.
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http://dx.doi.org/10.1126/scitranslmed.aay7675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7860587PMC
August 2020

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

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

Blood 2018 05 2;131(19):2097-2110. Epub 2018 Mar 2.

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

In the antiphospholipid syndrome (APS), antiphospholipid antibody (aPL) recognition of β2 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.
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http://dx.doi.org/10.1182/blood-2017-11-814681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946764PMC
May 2018

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

New Insights in the Pathophysiology of Antiphospholipid Syndrome.

Semin Thromb Hemost 2018 Jul 27;44(5):475-482. Epub 2017 Jan 27.

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

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.
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http://dx.doi.org/10.1055/s-0036-1597286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333209PMC
July 2018

Identification of a Monoclonal Antibody That Attenuates Antiphospholipid Syndrome-Related Pregnancy Complications and Thrombosis.

PLoS One 2016 27;11(7):e0158757. Epub 2016 Jul 27.

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

In the antiphospholipid syndrome (APS), patients produce antiphospholipid antibodies (aPL) that promote thrombosis and adverse pregnancy outcomes. Current therapy with anticoagulation is only partially effective and associated with multiple complications. We previously discovered that aPL recognition of cell surface β2-glycoprotein I (β2-GPI) initiates apolipoprotein E receptor 2 (apoER2)-dependent signaling in endothelial cells and in placental trophoblasts that ultimately promotes thrombosis and fetal loss, respectively. Here we sought to identify a monoclonal antibody (mAb) to β2-GPI that negates aPL-induced processes in cell culture and APS disease endpoints in mice. In a screen measuring endothelial NO synthase (eNOS) activity in cultured endothelial cells, we found that whereas aPL inhibit eNOS, the mAb 1N11 does not, and instead 1N11 prevents aPL action. Coimmunoprecipitation studies revealed that 1N11 decreases pathogenic antibody binding to β2-GPI, and it blocks aPL-induced complex formation between β2-GPI and apoER2. 1N11 also prevents aPL antagonism of endothelial cell migration, and in mice it reverses the impairment in reendothelialization caused by aPL, which underlies the non-thrombotic vascular occlusion provoked by disease-causing antibodies. In addition, aPL inhibition of trophoblast proliferation and migration is negated by 1N11, and the more than 6-fold increase in fetal resorption caused by aPL in pregnant mice is prevented by 1N11. Furthermore, the promotion of thrombosis by aPL is negated by 1N11. Thus, 1N11 has been identified as an mAb that attenuates APS-related pregnancy complications and thrombosis in mice. 1N11 may provide an efficacious, mechanism-based therapy to combat the often devastating conditions suffered by APS patients.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158757PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4963039PMC
July 2017

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

ApoE Receptor 2 Mediation of Trophoblast Dysfunction and Pregnancy Complications Induced by Antiphospholipid Antibodies in Mice.

Arthritis Rheumatol 2016 Mar;68(3):730-739

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

Objective: Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine whether the low-density lipoprotein receptor family member apolipoprotein E receptor 2 (ApoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL.

Methods: Placental and trophoblast ApoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG and aPL were purified from healthy individuals and APS patients, respectively. The role of ApoER2 in aPL-induced changes in trophoblast proliferation and migration and in kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of ApoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant ApoER2(+/+) and ApoER2(-/-) mice injected with aPL or normal human IgG.

Results: We found that ApoER2 is abundant in human and mouse placental trophoblasts and in multiple trophoblast-derived cell lines, including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2 -glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by epidermal growth factor in trophoblasts was mediated by ApoER2. Furthermore, in a murine passive-transfer model of pregnancy complications of APS, ApoER2(-/-) mice were protected from both aPL-induced fetal loss and aPL-induced IUGR.

Conclusion: ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS.
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http://dx.doi.org/10.1002/art.39453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4767551PMC
March 2016

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

Mol Cell 2015 Jun 14;58(5):845-53. Epub 2015 May 14.

Department of Dermatology, UT Southwestern Medical Center, Dallas, TX 75390, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.molcel.2015.04.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458224PMC
June 2015

PDZK1 prevents neointima formation via suppression of breakpoint cluster region kinase in vascular smooth muscle.

PLoS One 2015 17;10(4):e0124494. Epub 2015 Apr 17.

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

Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4401672PMC
April 2016

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

J Biol Chem 2012 Feb 10;287(9):6582-91. Epub 2012 Jan 10.

Division of Cardiology, Harvard Medical School, Boston, Massachusetts 02215, USA.

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.
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http://dx.doi.org/10.1074/jbc.M111.300236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3307294PMC
February 2012

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

Cells Tissues Organs 2012 13;195(1-2):122-43. Epub 2011 Oct 13.

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Dalton Cardiovascular Research Center, Columbia, Mo. 65212, USA.

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 α2β1, 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.
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http://dx.doi.org/10.1159/000331410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3325603PMC
April 2012

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

Blood 2011 Jul 31;118(4):1145-53. Epub 2011 May 31.

Division of Cardiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

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.
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http://dx.doi.org/10.1182/blood-2010-10-315275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3148162PMC
July 2011

Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice.

J Clin Invest 2011 May 1;121(5):1871-81. Epub 2011 Apr 1.

Molecular Medicine Program, University of Utah, Salt Lake City, Utah, USA.

Cerebral cavernous malformations (CCMs) are a common type of vascular malformation in the brain that are a major cause of hemorrhagic stroke. This condition has been independently linked to 3 separate genes: Krev1 interaction trapped (KRIT1), Cerebral cavernous malformation 2 (CCM2), and Programmed cell death 10 (PDCD10). Despite the commonality in disease pathology caused by mutations in these 3 genes, we found that the loss of Pdcd10 results in significantly different developmental, cell biological, and signaling phenotypes from those seen in the absence of Ccm2 and Krit1. PDCD10 bound to germinal center kinase III (GCKIII) family members, a subset of serine-threonine kinases, and facilitated lumen formation by endothelial cells both in vivo and in vitro. These findings suggest that CCM may be a common tissue manifestation of distinct mechanistic pathways. Nevertheless, loss of heterozygosity (LOH) for either Pdcd10 or Ccm2 resulted in CCMs in mice. The murine phenotype induced by loss of either protein reproduced all of the key clinical features observed in human patients with CCM, as determined by direct comparison with genotype-specific human surgical specimens. These results suggest that CCM may be more effectively treated by directing therapies based on the underlying genetic mutation rather than treating the condition as a single clinical entity.
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http://dx.doi.org/10.1172/JCI44393DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083782PMC
May 2011

Molecular basis for endothelial lumen formation and tubulogenesis during vasculogenesis and angiogenic sprouting.

Int Rev Cell Mol Biol 2011 ;288:101-65

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, Missouri, USA.

Many studies reveal a fundamental role for extracellular matrix-mediated signaling through integrins and Rho GTPases as well as matrix metalloproteinases (MMPs) in the molecular control of vascular tube morphogenesis in three-dimensional (3D) tissue environments. Recent work has defined an endothelial cell (EC) lumen signaling complex of proteins that controls these vascular morphogenic events. These findings reveal a signaling interdependence between Cdc42 and MT1-MMP to control the 3D matrix-specific process of EC tubulogenesis. The EC tube formation process results in the creation of a network of proteolytically generated vascular guidance tunnels in 3D matrices that are utilized to remodel EC-lined tubes through EC motility and could facilitate processes such as flow-induced remodeling and arteriovenous EC sorting and differentiation. Within vascular guidance tunnels, key dynamic interactions occur between ECs and pericytes to affect vessel remodeling, diameter, and vascular basement membrane matrix assembly, a fundamental process necessary for endothelial tube maturation and stabilization. Thus, the EC lumen and tube formation mechanism coordinates the concomitant establishment of a network of vascular tubes within tunnel spaces to allow for flow responsiveness, EC-mural cell interactions, and vascular extracellular matrix assembly to control the development of the functional microcirculation.
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http://dx.doi.org/10.1016/B978-0-12-386041-5.00003-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891664PMC
July 2011

Blood vessel tubulogenesis requires Rasip1 regulation of GTPase signaling.

Dev Cell 2011 Apr 10;20(4):526-39. Epub 2011 Mar 10.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Cardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However, the mechanisms underlying vascular "tubulogenesis" are only beginning to be unraveled. We show that endothelial tubulogenesis requires the Ras interacting protein 1, Rasip1, and its binding partner, the RhoGAP Arhgap29. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Similarly, depletion of either Rasip1 or Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton, and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. This study identifies Rasip1 as a unique, endothelial-specific regulator of Rho GTPase signaling, which is essential for blood vessel morphogenesis.
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http://dx.doi.org/10.1016/j.devcel.2011.02.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3078994PMC
April 2011

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

Blood 2010 Jun 9;115(25):5259-69. Epub 2010 Mar 9.

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, MO 85212, USA.

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.
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http://dx.doi.org/10.1182/blood-2009-11-252692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2892954PMC
June 2010

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

J Cell Sci 2009 Dec 24;122(Pt 24):4558-69. Epub 2009 Nov 24.

Department of Medical Pharmacology and Physiology, University of Missouri School of Medicine, Columbia, MO 65212, USA.

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.
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http://dx.doi.org/10.1242/jcs.050724DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2787465PMC
December 2009

Formation of endothelial lumens requires a coordinated PKCepsilon-, Src-, Pak- and Raf-kinase-dependent signaling cascade downstream of Cdc42 activation.

J Cell Sci 2009 Jun 12;122(Pt 11):1812-22. Epub 2009 May 12.

Department of Medical Pharmacology and Physiology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.

In this study, we present data showing that Cdc42-dependent lumen formation by endothelial cells (ECs) in three-dimensional (3D) collagen matrices involves coordinated signaling by PKCepsilon in conjunction with the Src-family kinases (SFKs) Src and Yes. Activated SFKs interact with Cdc42 in multiprotein signaling complexes that require PKCepsilon during this process. Src and Yes are differentially expressed during EC lumen formation and siRNA suppression of either kinase, but not Fyn or Lyn, results in significant inhibition of EC lumen formation. Concurrent with Cdc42 activation, PKCepsilon- and SFK-dependent signaling converge to activate p21-activated kinase (Pak)2 and Pak4 in steps that are also required for EC lumen formation. Pak2 and Pak4 further activate two Raf kinases, B-Raf and C-Raf, leading to ERK1 and ERK2 (ERK1/2) activation, which all seem to be necessary for EC lumen formation. This work reveals a multicomponent kinase signaling pathway downstream of integrin-matrix interactions and Cdc42 activation involving PKCepsilon, Src, Yes, Pak2, Pak4, B-Raf, C-Raf and ERK1/2 to control EC lumen formation in 3D collagen matrices.
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http://dx.doi.org/10.1242/jcs.045799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2684834PMC
June 2009

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

Blood 2009 Jul 1;114(2):237-47. Epub 2009 Apr 1.

Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Center, University of Missouri-Columbia, MO 65212, USA.

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.
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http://dx.doi.org/10.1182/blood-2008-12-196451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2714200PMC
July 2009

In vitro three dimensional collagen matrix models of endothelial lumen formation during vasculogenesis and angiogenesis.

Methods Enzymol 2008 ;443:83-101

Department of Medical Pharmacology and Physiology, School of Medicine, Dalton Cardiovascular Center, University of Missouri-Columbia, Columbia, Missouri, USA.

Discovery and comprehension of detailed molecular signaling pathways underlying endothelial vascular morphogenic events including endothelial lumen formation are key steps in understanding their roles during embryonic development, as well as during various disease states. Studies that used in vitro three-dimensional (3D) matrix endothelial cell morphogenic assay models, in conjunction with in vivo studies, have been essential to identifying molecules and explaining their related signaling pathways that regulate endothelial cell morphogenesis. We present methods to study molecular mechanisms controlling EC lumen formation in 3D collagen matrices. In vitro models representing vasculogenesis and angiogenesis, whereby EC lumen formation and tube morphogenesis readily occur, are described. We also detail different methods of gene manipulation in ECs and their application to analyze critical signaling events regulating EC lumen formation.
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http://dx.doi.org/10.1016/S0076-6879(08)02005-3DOI Listing
January 2009

Determinants for association and guide RNA-directed endonuclease cleavage by purified RNA editing complexes from Trypanosoma brucei.

J Mol Biol 2008 Aug 8;381(1):35-48. Epub 2008 May 8.

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843, USA.

U-insertion/deletion RNA editing in the single mitochondrion of kinetoplastids, an ancient lineage of eukaryotes, is a unique mRNA maturation process needed for translation. Multisubunit editing complexes recognize many pre-edited mRNA sites and modify them via cycles of three catalytic steps: guide RNA (gRNA)-directed cleavage, insertion or deletion of uridylates at the 3'-terminus of the upstream cleaved piece, and ligation of the two mRNA pieces. While catalytic and many structural protein subunits of these complexes have been identified, the mechanisms and basic determinants of substrate recognition are still poorly understood. This study defined relatively simple single- and double-stranded determinants for association and gRNA-directed cleavage. To this end, we used an electrophoretic mobility shift assay to directly score the association of purified editing complexes with RNA ligands, in parallel with UV photocrosslinking and functional studies. The cleaved strand required a minimal 5' overhang of 12 nt and an approximately 15-bp duplex with gRNA to direct the cleavage site. A second protruding element in either the cleaved or the guide strand was required unless longer duplexes were used. Importantly, the single-stranded RNA requirement for association can be upstream or downstream of the duplex, and the binding and cleavage activities of purified editing complexes could be uncoupled. The current observations together with our previous reports in the context of purified native editing complexes show that the determinants for association, cleavage and full-round editing gradually increase in complexity as these stages progress. The native complexes in these studies contained most, if not all, known core subunits in addition to components of the MRP complex. Finally, we found that the endonuclease KREN1 in purified complexes photocrosslinks with a targeted editing site. A model is proposed whereby one or more RNase III-type endonucleases mediate the initial binding and scrutiny of potential ligands and subsequent catalytic selectivity triggers either insertion or deletion editing enzymes.
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http://dx.doi.org/10.1016/j.jmb.2008.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2596986PMC
August 2008

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

Int J Parasitol 2006 Oct 19;36(12):1295-304. Epub 2006 Jun 19.

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843, USA.

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.
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http://dx.doi.org/10.1016/j.ijpara.2006.05.011DOI Listing
October 2006

RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei.

RNA 2006 Jul 11;12(7):1219-28. Epub 2006 May 11.

Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA.

Trypanosome U insertion and U deletion RNA editing of mitochondrial pre-mRNAs is catalyzed by multisubunit editing complexes as directed by partially complementary guide RNAs. The basic enzymatic activities and protein composition of these high-molecular mass complexes have been under intense study, but their specific protein interactions with functional pre-mRNA/gRNA substrates remains unknown. We show that editing complexes purified through extensive ion-exchange chromatography and immunoprecipitation make specific cross-linking interactions with A6 pre-mRNA containing a single 32P and photoreactive 4-thioU at the scissile bond of a functional site for full-round U deletion. At least four direct protein-RNA contacts are detected at this site by cross-linking. All four interactions are stimulated by unpaired residues just 5' of the pre-mRNA/gRNA anchor duplex, but strongly inhibited by pairing of the editing site region. Furthermore, competition analysis with homologous and heterologous transcripts suggests preferential contacts of the editing complex with the mRNA/gRNA duplex substrate. This apparent structural selectivity suggests that the RNA-protein interactions we observe may be involved in recognition of editing sites and/or catalysis in assembled complexes.
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http://dx.doi.org/10.1261/rna.2295706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1484423PMC
July 2006

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

Nucleic Acids Res 2005 23;33(20):6610-20. Epub 2005 Nov 23.

Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843, USA.

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.
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http://dx.doi.org/10.1093/nar/gki943DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1298919PMC
December 2005