Platelet Surface PDI Controls the Ligand-Binding Function of Glycoprotein Ibalpha and Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 235-235
Author(s):  
Kyungho Kim ◽  
Jing Li ◽  
Robert K Andrews ◽  
Joyce Chiu ◽  
Philip Hogg ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Disulfide Bond ◽  
Conflicts Of Interest ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Conditional Knockout ◽  
Spectrometric Analysis ◽  
Platelet Surface ◽  
Isomerase Activity ◽  
Binding Function

Abstract We previously showed that the isomerase activity of platelet surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, regulates the full activation of αIIbβ3 integrin and is important for platelet accumulation, but not hemostasis, following vascular injury (Kim et al. Blood 2013). However, it remains unclear whether platelet PDI regulates the function of other platelet surface receptors. Since it was reported that platelet PDI and glycoprotein Ibα (GPIbα) were physically close on the platelet surface (Burgess et al. JBC 2000), we investigated whether platelet PDI regulates GPIbα function using megakaryocyte-specific PDI conditional knockout (CKO) mice developed by us. We found that PDI-null platelets showed a significant defect in GPIbα-mediated agglutination and von Willebrand factor (vWF) binding. Those defects were completely rescued by exogenously-added recombinant wild-type PDI (wtPDI), but not mutant PDI (dmPDI) lacking the isomerase activity. Consistently, inhibition of platelet PDI with blocking antibodies impaired GPIbα-mediated agglutination and vWF binding in human and mouse platelets, suggesting that the isomerase activity of platelet surface PDI controls the ligand-binding function of GPIbα. Studies using surface plasmon resonance revealed that wtPDI and dmPDI bound to immobilized GPIbα with a dissociation constant of 0.9 and 1.2 µM, respectively. Mass spectrometric analysis indicated that the Cys4-Cys17 disulfide bond in GPIbα, which has an allosteric -RHStaple configuration, was preferentially reduced by wtPDI, but not dmPDI. Consistent with previous studies showing that platelet GPIbα is important for platelet-neutrophil interactions, we observed that deletion of platelet PDI significantly reduced platelet-neutrophil interactions under shear conditions. Importantly, using real-time fluorescence intravital microscopy with megakaryocyte-specific PDI CKO mice, we demonstrated that platelet-specific PDI deletion did not influence neutrophil adhesion but markedly reduced platelet adhesion and accumulation on the adherent neutrophils on TNF-α-inflamed cremaster muscle venules. Infusion of wtPDI, but not dmPDI, restored reduced platelet-neutrophil interactions in the PDI CKO mice, implying the importance of platelet surface PDI activity in regulating GPIbα-mediated platelet-neutrophil interactions. Using a thromboinflammation model of hepatic ischemia/reperfusion injury, we found that platelet-specific PDI CKO mice exhibited a significant reduction in the infarct size and the serum levels of aspartate aminotransferase, a marker of liver damage. Our results suggest that the isomerase activity of platelet surface PDI regulates the redox state of the Cys4-Cys17 disulfide bond in GPIbα and is required for the ligand-binding function of GPIbα, thereby playing a critical role during platelet-neutrophil interactions under thromboinflammatory conditions. Disclosures No relevant conflicts of interest to declare.

PRMT5 Methyltransferase Activity Is Required to Sustain Adult Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4335-4335
Author(s):  
Fan LIU ◽  
Guoyan Cheng ◽  
Fabiana Perna ◽  
Xu Haiming ◽  
Pierre-Jacques Hamard ◽  
...  
Keyword(s):  
Cell Biology ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Conditional Knockout ◽  
Major Type ◽  
Cytokine Signaling ◽  
Methyltransferase Activity ◽  
Hematopoietic Stem ◽  
Arginine Residues

Abstract Epigenetic regulators have been shown to play critical roles in normal hematopoiesis, and their activity is frequently altered in hematopoietic cancers. Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMTs, catalyzing the symmetric di-methylation of arginine residues in histones (H2A, H3 and H4) and non-histone proteins. PRMT5 is over-expressed in several cancers, including acute leukemia and non-Hodgkin’s lymphoma. To define the role of PRMT5 in normal adult hematopoiesis, we generated PRMT5 conditional knockout mice using Mx1-cre. The induced deletion of both alleles of PRMT5 leads to severe pancytopenia and bone marrow aplasia with subsequent lethality in two weeks. First, loss of PRMT5 triggers the impaired proliferation and rapid disappearance of progenitor cells. At the same time, PRMT5 deficient HSCs show increased cell cycling and a transient HSC accumulation, which is rapidly followed by stem cell exhaustion. Mechanistically, we show that deletion of PRMT5 severely impairs cytokine signaling. It also up-regulates p53 protein level and the expression of p53 target genes. These effects likely account for the critical role of PRMT5 in HSPCs. We have conducted many additional experiments to show that these effects of PRMT5 deletion on hematopoiesis are cell autonomous; and also that the methyltransferase activity of PRMT5 is required to sustain normal hematopoiesis. Thus, we identify PRMT5 as a critical regulator of normal hematopoietic stem and progenitor cell biology. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sequential activation of necroptosis and apoptosis cooperates to mediate vascular and neural pathology in stroke

2020 ◽  
Vol 117 (9) ◽  
pp. 4959-4970 ◽  
Author(s):  
Masanori Gomi Naito ◽  
Daichao Xu ◽  
Palak Amin ◽  
Jinwoo Lee ◽  
Huibing Wang ◽  
...  
Keyword(s):  
Cell Death ◽  
Cerebral Ischemia ◽  
Cerebral Hemorrhage ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Arterial Occlusion ◽  
Conditional Knockout ◽  
Cerebral Ischemia Reperfusion ◽  
Cerebral Pathology

Apoptosis and necroptosis are two regulated cell death mechanisms; however, the interaction between these cell death pathways in vivo is unclear. Here we used cerebral ischemia/reperfusion as a model to investigate the interaction between apoptosis and necroptosis. We show that the activation of RIPK1 sequentially promotes necroptosis followed by apoptosis in a temporally specific manner. Cerebral ischemia/reperfusion insult rapidly activates necroptosis to promote cerebral hemorrhage and neuroinflammation.Ripk3deficiency reduces cerebral hemorrhage and delays the onset of neural damage mediated by inflammation. Reduced cerebral perfusion resulting from arterial occlusion promotes the degradation of TAK1, a suppressor of RIPK1, and the transition from necroptosis to apoptosis. Conditional knockout of TAK1 in microglial/infiltrated macrophages and neuronal lineages sensitizes to ischemic infarction by promoting apoptosis. Taken together, our results demonstrate the critical role of necroptosis in mediating neurovascular damage and hypoperfusion-induced TAK1 loss, which subsequently promotes apoptosis and cerebral pathology in stroke and neurodegeneration.

scholarly journals LAMTOR2 Is Dispensable for T Cell Receptor-Mediated Signaling of Thymocytes but Controls Medullary Thymic Epithelial Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1122-1122
Author(s):  
Lina Wendeler ◽  
Lukas A Huber ◽  
Christoph Klein ◽  
Daniel Kotlarz
Keyword(s):  
T Cell ◽  
Epithelial Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
T Cell Development ◽  
Cell Development ◽  
Conditional Knockout ◽  
Poly I:C ◽  
Thymic Epithelial Cells

Abstract Human LAMTOR2 deficiency is characterized by severe congenital neutropenia, growth failure, partial albinism, as well as B and T cell deficiencies (Bohn et al., Nat Med 2007). To determine the role of the endosomal adaptor LAMTOR2 in T cell development and homeostasis we used conditional knockout mouse models. Mx1-Cre-driven knockout of Lamtor2 resulted in reduction of thymus weight and total thymocyte numbers. Immunophenotyping revealed an impaired T cell development characterized by a partial block at the double negative CD4-CD8- T cell precursor stage after 7 and 21 days of poly I:C injection that induced deletion of the Lamtor2 gene. Since Mx1-Cre-driven knockout does not allow a discrimination between T cell intrinsic and extrinsic effects, we next generated pre-TCRα-iCre conditional knockout mice. In contrast to Mx1-Cre-Lamtor2fl/fl mice, mice with T cell-specific knockout of Lamtor2 showed normal frequencies of total thymocytes and T cell progenitor subsets. Furthermore, LAMTOR2-deficient thymocytes exhibited normal TCR signaling (p-ERK, p-LAT, p-LCK, p-PLCγ, Nur77) and internalization of TCRβ upon stimulation with anti-CD3ε +/- anti-CD28, indicating that LAMTOR2 in T cells is dispensable for thymocyte development. To assess whether T cell developmental defects in Mx1-Cre-Lamtor2fl/fl mice are caused by a dysfunctional thymic epithelium, we analyzed thymic epithelial cells (TECs) after 4 days of poly I:C injection by flow cytometry and detected a reduced ratio of CD45-EpCAM+UEA-1+Ly51- medullary TECs (mTECs) to CD45-EpCAM+UEA-1-Ly51+ cortical TECs in LAMTOR2-deficient mice. Further studies are underway to determine the role of LAMTOR2 in mTECs. Taken together, our findings show that LAMTOR2 is not required for TCR-mediated signaling but plays a critical role in controlling mTEC homeostasis. Disclosures No relevant conflicts of interest to declare.

LncRNA SNHG12 Improves Cerebral Ischemic-reperfusion Injury by Activating SIRT1/FOXO3a Pathway through I nhibition of Autophagy and Oxidative Stress

2020 ◽  
Vol 17 (4) ◽  
pp. 394-401
Author(s):  
Yuanhua Wu ◽  
Yuan Huang ◽  
Jing Cai ◽  
Donglan Zhang ◽  
Shixi Liu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Molecular Mechanisms ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Cell Activity ◽  
Ht22 Cells ◽  
Cerebral Ischemic ◽  
Cerebral Ischemic Reperfusion ◽  
And Oxidative Stress

Background: Ischemia/reperfusion (I/R) injury involves complex biological processes and molecular mechanisms such as autophagy. Oxidative stress plays a critical role in the pathogenesis of I/R injury. LncRNAs are the regulatory factor of cerebral I/R injury. Methods: This study constructs cerebral I/R model to investigate role of autophagy and oxidative stress in cerebral I/R injury and the underline regulatory mechanism of SIRT1/ FOXO3a pathway. In this study, lncRNA SNHG12 and FOXO3a expression was up-regulated and SIRT1 expression was down-regulated in HT22 cells of I/R model. Results: Overexpression of lncRNA SNHG12 significantly increased the cell viability and inhibited cerebral ischemicreperfusion injury induced by I/Rthrough inhibition of autophagy. In addition, the transfected p-SIRT1 significantly suppressed the release of LDH and SOD compared with cells co-transfected with SIRT1 and FOXO3a group and cells induced by I/R and transfected with p-SNHG12 group and overexpression of cells co-transfected with SIRT1 and FOXO3 further decreased the I/R induced release of ROS and MDA. Conclusion: In conclusion, lncRNA SNHG12 increased cell activity and inhibited oxidative stress through inhibition of SIRT1/FOXO3a signaling-mediated autophagy in HT22 cells of I/R model. This study might provide new potential therapeutic targets for further investigating the mechanisms in cerebral I/R injury and provide.

KAUTILYA ON THE SCOPE AND METHODOLOGY OF ACCOUNTING, ORGANIZATIONAL DESIGN AND THE ROLE OF ETHICS IN ANCIENT INDIA

2004 ◽  
Vol 31 (2) ◽  
pp. 125-148 ◽  
Author(s):  
Balbir S. Sihag
Keyword(s):  
Economic Development ◽  
Economic Performance ◽  
Organizational Design ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ethical Values ◽  
Allocation Of Resources ◽  
Economic Data ◽  
Ancient India

Kautilya, a 4th century B.C.E. economist, recognized the importance of accounting methods in economic enterprises. He realized that a proper measurement of economic performance was absolutely essential for efficient allocation of resources, which was considered an important source of economic development. He viewed philosophy and political science as separate disciplines but considered accounting an integral part of economics. He specified a very broad scope for accounting and considered explanation and prediction as its proper objectives. Kautilya developed bookkeeping rules to record and classify economic data, emphasized the critical role of independent periodic audits and proposed the establishment of two important but separate offices - the Treasurer and Comptroller-Auditor, to increase accountability, specialization, and above all to reduce the scope for conflicts of interest. He also linked the successful enforcement of rules and regulations to their clarity, consistency and completeness. Kautilya believed that such measures were necessary but not sufficient to eliminate fraudulent accounting. He also emphasized the role of ethics, considering ethical values as the glue which binds society and promotes economic development.

scholarly journals Mitochondrial fission and mitophagy are independent mechanisms regulating ischemia/reperfusion injury in primary neurons

2021 ◽  
Vol 12 (5) ◽  
Author(s):  
Anthony R. Anzell ◽  
Garrett M. Fogo ◽  
Zoya Gurm ◽  
Sarita Raghunayakula ◽  
Joseph M. Wider ◽  
...  
Keyword(s):  
Cortical Neurons ◽  
Ischemia Reperfusion Injury ◽  
Mitochondrial Dynamics ◽  
Ischemia Reperfusion ◽  
Mitochondrial Fission ◽  
Conditional Knockout ◽  
Mitochondrial Morphology ◽  
Primary Neurons ◽  
Mitochondrial Fragmentation

AbstractMitochondrial dynamics and mitophagy are constitutive and complex systems that ensure a healthy mitochondrial network through the segregation and subsequent degradation of damaged mitochondria. Disruption of these systems can lead to mitochondrial dysfunction and has been established as a central mechanism of ischemia/reperfusion (I/R) injury. Emerging evidence suggests that mitochondrial dynamics and mitophagy are integrated systems; however, the role of this relationship in the context of I/R injury remains unclear. To investigate this concept, we utilized primary cortical neurons isolated from the novel dual-reporter mitochondrial quality control knockin mice (C57BL/6-Gt(ROSA)26Sortm1(CAG-mCherry/GFP)Ganl/J) with conditional knockout (KO) of Drp1 to investigate changes in mitochondrial dynamics and mitophagic flux during in vitro I/R injury. Mitochondrial dynamics was quantitatively measured in an unbiased manner using a machine learning mitochondrial morphology classification system, which consisted of four different classifications: network, unbranched, swollen, and punctate. Evaluation of mitochondrial morphology and mitophagic flux in primary neurons exposed to oxygen-glucose deprivation (OGD) and reoxygenation (OGD/R) revealed extensive mitochondrial fragmentation and swelling, together with a significant upregulation in mitophagic flux. Furthermore, the primary morphology of mitochondria undergoing mitophagy was classified as punctate. Colocalization using immunofluorescence as well as western blot analysis revealed that the PINK1/Parkin pathway of mitophagy was activated following OGD/R. Conditional KO of Drp1 prevented mitochondrial fragmentation and swelling following OGD/R but did not alter mitophagic flux. These data provide novel evidence that Drp1 plays a causal role in the progression of I/R injury, but mitophagy does not require Drp1-mediated mitochondrial fission.

scholarly journals Ginsenoside Rb1 Treatment Attenuates Pulmonary Inflammatory Cytokine Release and Tissue Injury following Intestinal Ischemia Reperfusion Injury in Mice

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Ying Jiang ◽  
Zhen Zhou ◽  
Qing-tao Meng ◽  
Qian Sun ◽  
Wating Su ◽  
...  
Keyword(s):  
Lung Injury ◽  
Signaling Pathway ◽  
Intestinal Ischemia ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Weight Ratio ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Ginsenoside Rb1 ◽  
Intestinal Ischemia Reperfusion

Objective. Intestinal ischemia reperfusion (II/R) injury plays a critical role in remote organ dysfunction, such as lung injury, which is associated with nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. In the present study, we tested whether ginsenoside Rb1 attenuated II/R induced lung injury by Nrf2/HO-1 pathway.Methods. II/R injury was induced in male C57BL/6J mice by 45 min of superior mesenteric artery (SMA) occlusion followed by 2 hours of reperfusion. Ginsenoside Rb1 was administrated prior to reperfusion with or without ATRA (all-transretinoic acid, the inhibitor of Nrf2/ARE signaling pathway) administration before II/R.Results. II/R induced lung histological injury, which is accompanied with increased levels of malondialdehyde (MDA), interleukin- (IL-) 6, and tumor necrosis factor- (TNF-)αbut decreased levels of superoxide dismutase (SOD) and IL-10 in the lung tissues. Ginsenoside Rb1 reduced lung histological injury and the levels of TNF-αand MDA, as well as wet/dry weight ratio. Interestingly, the increased Nrf2 and HO-1 expression induced by II/R in the lung tissues was promoted by ginsenoside Rb1 treatment. All these changes could be inhibited or prevented by ATRA.Conclusion. Ginsenoside Rb1 is capable of ameliorating II/R induced lung injuries by activating Nrf2/HO-1 pathway.

scholarly journals Brain-Specific SNAP-25 Deletion Leads to Elevated Extracellular Glutamate Level and Schizophrenia-Like Behavior in Mice

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Hua Yang ◽  
Mengjie Zhang ◽  
Jiahao Shi ◽  
Yunhe Zhou ◽  
Zhipeng Wan ◽  
...  
Keyword(s):  
Cerebral Cortex ◽  
Mouse Model ◽  
Glutamate Release ◽  
Critical Role ◽  
Conditional Knockout ◽  
Snare Complex ◽  
Extracellular Glutamate ◽  
Glutamate Level

Several studies have associated reduced expression of synaptosomal-associated protein of 25 kDa (SNAP-25) with schizophrenia, yet little is known about its role in the illness. In this paper, a forebrain glutamatergic neuron-specific SNAP-25 knockout mouse model was constructed and studied to explore the possible pathogenetic role of SNAP-25 in schizophrenia. We showed that SNAP-25 conditional knockout (cKO) mice exhibited typical schizophrenia-like phenotype. A significantly elevated extracellular glutamate level was detected in the cerebral cortex of the mouse model. Compared with Ctrls, SNAP-25 was dramatically reduced by about 60% both in cytoplasm and in membrane fractions of cerebral cortex of cKOs, while the other two core members of SNARE complex: Syntaxin-1 (increased ~80%) and Vamp2 (increased ~96%) were significantly increased in cell membrane part. Riluzole, a glutamate release inhibitor, significantly attenuated the locomotor hyperactivity deficits in cKO mice. Our findings provide in vivo functional evidence showing a critical role of SNAP-25 dysfunction on synaptic transmission, which contributes to the developmental of schizophrenia. It is suggested that a SNAP-25 cKO mouse, a valuable model for schizophrenia, could address questions regarding presynaptic alterations that contribute to the etiopathophysiology of SZ and help to consummate the pre- and postsynaptic glutamatergic pathogenesis of the illness.

Sign in / Sign up

Export Citation Format

Share Document