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The Journal of Clinical Investigation Jun 2023Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This...
Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases," such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.
Topics: Animals; Mice; Blood Platelets; Cardiovascular Diseases; Endothelial Cells; Mice, Knockout; Phosphatidylserines; Phospholipid Transfer Proteins; Phospholipids; Thrombosis
PubMed: 36951953
DOI: 10.1172/JCI163808 -
Theranostics 2021Ischemic stroke remains a major cause of death, and anti-inflammatory strategies hold great promise for preventing major brain injury during reperfusion. In the past...
Ischemic stroke remains a major cause of death, and anti-inflammatory strategies hold great promise for preventing major brain injury during reperfusion. In the past decade, stem cell-derived extracellular vesicles (EVs) have emerged as novel therapeutic effectors in immune modulation. However, the intravenous delivery of EVs into the ischemic brain remains a challenge due to poor targeting of unmodified EVs, and the costs of large-scale production of stem cell-derived EVs hinder their clinical application. EVs were isolated from a human neural progenitor cell line, and their anti-inflammatory effects were verified . To attach targeting ligands onto EVs, we generated a recombinant fusion protein containing the arginine-glycine-aspartic acid (RGD)-4C peptide (ACDCRGDCFC) fused to the phosphatidylserine (PS)-binding domains of lactadherin (C1C2), which readily self-associates onto the EV membrane. Subsequently, in a middle cerebral artery occlusion (MCAO) mouse model, the RGD-C1C2-bound EVs (RGD-EV) were intravenously injected through the tail vein, followed by fluorescence imaging and assessment of proinflammatory cytokines expression and microglia activation. The neural progenitor cell-derived EVs showed intrinsic anti-inflammatory activity. The RGD-EV targeted the lesion region of the ischemic brain after intravenous administration, and resulted in a strong suppression of the inflammatory response. Furthermore, RNA sequencing revealed a set of 7 miRNAs packaged in the EVs inhibited MAPK, an inflammation related pathway. These results point to a rapid and easy strategy to produce targeting EVs and suggest a potential therapeutic agent for ischemic stroke.
Topics: Animals; Antigens, Surface; Brain Ischemia; Cells, Cultured; Culture Media, Conditioned; Extracellular Vesicles; Genes, Reporter; HEK293 Cells; Humans; Infarction, Middle Cerebral Artery; Inflammation; Injections, Intravenous; Lipopolysaccharides; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; MicroRNAs; Microglia; Milk Proteins; Nanoparticles; Neural Stem Cells; Oligopeptides; Phosphatidylserines; Recombinant Fusion Proteins
PubMed: 33995671
DOI: 10.7150/thno.56367 -
Immunity Aug 2020CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple...
CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.
Topics: Animals; CD47 Antigen; Calreticulin; Cell Line; Complement System Proteins; HEK293 Cells; Humans; Immunoglobulin G; Integrins; Macrophage Activation; Macrophages; Mice; Mice, Inbred C57BL; Phagocytosis; Phosphatidylserines; RAW 264.7 Cells; Receptors, Immunologic; Signal Transduction
PubMed: 32768386
DOI: 10.1016/j.immuni.2020.07.008 -
Cellular Immunology Feb 2023Phosphatidylserine (PS) is an anionic phospholipid exposed on the surface of apoptotic cells. The exposure of PS typically recruits and signals phagocytes to engulf and...
Phosphatidylserine (PS) is an anionic phospholipid exposed on the surface of apoptotic cells. The exposure of PS typically recruits and signals phagocytes to engulf and silently clear these dying cells to maintain tolerance via immunological ignorance. However, recent and emerging evidence has demonstrated that PS converts an "immunogen" into a "tolerogen", and PS exposure on the surface of cells or vesicles actively promotes a tolerogenic environment. This tolerogenic property depends on the biophysical characteristics of PS-containing vesicles, including PS density on the particle surface to effectively engage tolerogenic receptors, such as TIM-4, which is exclusively expressed on the surface of antigen-presenting cells. We harnessed the cellular and molecular mechanistic insight of PS-mediated immune regulation to design an effective oral tolerance approach. This immunotherapy has been shown to prevent/reduce immune response against life-saving protein-based therapies, food allergens, autoantigens, and the antigenic viral capsid peptide commonly used in gene therapy, suggesting a broad spectrum of potential clinical applications. Given the good safety profile of PS together with the ease of administration, oral tolerance achieved with PS-based nanoparticles has a very promising therapeutic impact.
Topics: Phosphatidylserines; Immunotherapy; Antigen-Presenting Cells; Autoantigens; Immune Tolerance; Apoptosis
PubMed: 36586393
DOI: 10.1016/j.cellimm.2022.104660 -
American Journal of Physiology. Cell... Oct 2022Platelets play a key role in maintaining hemostasis. However, dysregulated platelet activation can lead to pathological thrombosis or bleeding. Once a platelet gets... (Review)
Review
Platelets play a key role in maintaining hemostasis. However, dysregulated platelet activation can lead to pathological thrombosis or bleeding. Once a platelet gets activated, it will either become an aggregatory platelet or eventually a procoagulant platelet with both types playing distinct roles in thrombosis and hemostasis. Although aggregatory platelets have been extensively studied, procoagulant platelets have only recently come into the spotlight. Procoagulant platelets are a subpopulation of highly activated platelets that express phosphatidylserine and P-selectin on their surface, allowing for coagulation factors to bind and thrombin to be generated. In recent years, novel roles for procoagulant platelets have been identified and they have increasingly been implicated in thromboinflammatory diseases. Here, we provide an up-to-date review on the mechanisms resulting in the formation of procoagulant platelets and how they contribute to hemostasis, thrombosis, and thromboinflammation.
Topics: Blood Coagulation; Blood Platelets; Humans; Inflammation; P-Selectin; Phosphatidylserines; Platelet Activation; Thrombin; Thromboinflammation; Thrombosis
PubMed: 35993516
DOI: 10.1152/ajpcell.00252.2022 -
Expert Reviews in Molecular Medicine Aug 2022Thrombosis is a common disorder with a relevant burden of morbidity and mortality worldwide, particularly among elderly patients. Growing evidence demonstrated a direct... (Review)
Review
Thrombosis is a common disorder with a relevant burden of morbidity and mortality worldwide, particularly among elderly patients. Growing evidence demonstrated a direct role of oxidative stress in thrombosis, with various cell types contributing to this process. Among them, erythrocytes produce high quantities of intracellular reactive oxygen species (ROS) by NADPH oxidase activation and haemoglobin autoxidation. Concomitantly, extracellular ROS released by other cells in the blood flow can be uptaken and accumulate within erythrocytes. This oxidative milieu can alter erythrocyte membrane structure, leading to an impaired erythrocyte function, and promoting erythrocytes lysis, binding to endothelial cells, activation of platelet and of coagulation factors, phosphatidylserine exposure and release of microvesicles. Moreover, these abnormal erythrocytes are able to adhere to the vessel wall, contributing to thrombin generation within the thrombus. This process results in accelerated haemolysis and in a hypercoagulable state, in which structurally impaired erythrocytes contribute to increase thrombus size, to reduce its permeability and susceptibility to lysis. However, the wide plethora of mechanisms by which oxidised erythrocytes contribute to thrombosis is not completely elucidated. This review discusses the main biochemical aspects linking erythrocytes, oxidative stress and thrombosis, addressing their potential implication for clinical and therapeutic management.
Topics: Aged; Endothelial Cells; Erythrocytes; Hemoglobins; Humans; NADPH Oxidases; Oxidative Stress; Phosphatidylserines; Reactive Oxygen Species; Thrombin; Thrombosis
PubMed: 36017709
DOI: 10.1017/erm.2022.25 -
Biochemical Society Transactions Oct 2022Phagocytosis triggered by the phospholipid phosphatidylserine (PS) is key for the removal of apoptotic cells in development, tissue homeostasis and infection. Modulation... (Review)
Review
Phagocytosis triggered by the phospholipid phosphatidylserine (PS) is key for the removal of apoptotic cells in development, tissue homeostasis and infection. Modulation of PS-mediated phagocytosis is an attractive target for therapeutic intervention in the context of atherosclerosis, neurodegenerative disease, and cancer. Whereas the mechanisms of target recognition, lipid and protein signalling, and cytoskeletal remodelling in opsonin-driven modes of phagocytosis are increasingly well understood, PS-mediated phagocytosis has remained more elusive. This is partially due to the involvement of a multitude of receptors with at least some redundancy in functioning, which complicates dissecting their contributions and results in complex downstream signalling networks. This review focusses on the receptors involved in PS-recognition, the signalling cascades that connect receptors to cytoskeletal remodelling required for phagocytosis, and recent progress in our understanding of how phagocytic cup formation is coordinated during PS-mediated phagocytosis.
Topics: Humans; Phosphatidylserines; Neurodegenerative Diseases; Apoptosis; Phagocytosis; Signal Transduction
PubMed: 36281986
DOI: 10.1042/BST20211254 -
Critical Reviews in Biochemistry and... Apr 2020P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and... (Review)
Review
P4-ATPases, a subfamily of P-type ATPases, translocate cell membrane phospholipids from the exoplasmic/luminal leaflet to the cytoplasmic leaflet to generate and maintain membrane lipid asymmetry. Exposure of phosphatidylserine (PS) in the exoplasmic leaflet is well known to transduce critical signals for apoptotic cell clearance and platelet coagulation. PS exposure is also involved in many other biological processes, including myoblast and osteoclast fusion, and the immune response. Moreover, mounting evidence suggest that PS exposure is critical for neuronal regeneration and degeneration. In apoptotic cells, PS exposure is induced by irreversible activation of scramblases and inactivation of P4-ATPases. However, how PS is reversibly exposed and restored in viable cells during other biological processes remains poorly understood. In the present review, we discuss the physiological significance of reversible PS exposure in living cells, and the putative roles of flippases, floppases, and scramblases.
Topics: Animals; Apoptosis; Cell Membrane; Cell Survival; Cytoplasm; Humans; Lipid Bilayers; P-type ATPases; Phosphatidylserines; Phospholipid Transfer Proteins; Platelet Activation; Substrate Specificity
PubMed: 32408772
DOI: 10.1080/10409238.2020.1758624 -
The Journal of Cell Biology Jun 2021TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins....
TMEM41B and VMP1 are integral membrane proteins of the endoplasmic reticulum (ER) and regulate the formation of autophagosomes, lipid droplets (LDs), and lipoproteins. Recently, TMEM41B was identified as a crucial host factor for infection by all coronaviruses and flaviviruses. The molecular function of TMEM41B and VMP1, which belong to a large evolutionarily conserved family, remains elusive. Here, we show that TMEM41B and VMP1 are phospholipid scramblases whose deficiency impairs the normal cellular distribution of cholesterol and phosphatidylserine. Their mechanism of action on LD formation is likely to be different from that of seipin. Their role in maintaining cellular phosphatidylserine and cholesterol homeostasis may partially explain their requirement for viral infection. Our results suggest that the proper sorting and distribution of cellular lipids are essential for organelle biogenesis and viral infection.
Topics: Autophagosomes; Autophagy; Cholesterol; Endoplasmic Reticulum; HeLa Cells; Humans; Lipid Droplets; Membrane Proteins; Phosphatidylserines; Protein Transport
PubMed: 33929485
DOI: 10.1083/jcb.202103105 -
The Journal of Biological Chemistry 2021Formations of myofibers, osteoclasts, syncytiotrophoblasts, and fertilized zygotes share a common step, cell-cell fusion. Recent years have brought about considerable... (Review)
Review
Formations of myofibers, osteoclasts, syncytiotrophoblasts, and fertilized zygotes share a common step, cell-cell fusion. Recent years have brought about considerable progress in identifying some of the proteins involved in these and other cell-fusion processes. However, even for the best-characterized cell fusions, we still do not know the mechanisms that regulate the timing of cell-fusion events. Are they fully controlled by the expression of fusogenic proteins or do they also depend on some triggering signal that activates these proteins? The latter scenario would be analogous to the mechanisms that control the timing of exocytosis initiated by Ca influx and virus-cell fusion initiated by low pH- or receptor interaction. Diverse cell fusions are accompanied by the nonapoptotic exposure of phosphatidylserine at the surface of fusing cells. Here we review data on the dependence of membrane remodeling in cell fusion on phosphatidylserine and phosphatidylserine-recognizing proteins and discuss the hypothesis that cell surface phosphatidylserine serves as a conserved "fuse me" signal regulating the time and place of cell-fusion processes.
Topics: Cell Fusion; Exocytosis; Humans; Phosphatidylserines; Signal Transduction; Virus Internalization
PubMed: 33581114
DOI: 10.1016/j.jbc.2021.100411