Publications by authors named "Changsen Wang"

17 Publications

  • Page 1 of 1

Endothelial HMGB1 Is a Critical Regulator of LDL Transcytosis via an SREBP2-SR-BI Axis.

Arterioscler Thromb Vasc Biol 2021 01 15;41(1):200-216. Epub 2020 Oct 15.

Keenan Centre for Biomedical Research, St. Michael's Hospital, Toronto, Canada (S.G., F.N.N., R.S., N.K., C.W., W.L.L.).

Objective: LDL (low-density lipoprotein) transcytosis across the endothelium is performed by the SR-BI (scavenger receptor class B type 1) receptor and contributes to atherosclerosis. HMGB1 (high mobility group box 1) is a structural protein in the nucleus that is released by cells during inflammation; extracellular HMGB1 has been implicated in advanced disease. Whether intracellular HMGB1 regulates LDL transcytosis through its nuclear functions is unknown. Approach and Results: HMGB1 was depleted by siRNA in human coronary artery endothelial cells, and transcytosis of LDL was measured by total internal reflection fluorescence microscopy. Knockdown of HMGB1 attenuated LDL transcytosis without affecting albumin transcytosis. Loss of HMGB1 resulted in reduction in SR-BI levels and depletion of SREBP2 (sterol regulatory element-binding protein 2)-a transcription factor upstream of SR-BI. The effect of HMGB1 depletion on LDL transcytosis required SR-BI and SREBP2. Overexpression of HMGB1 caused an increase in LDL transcytosis that was unaffected by inhibition of extracellular HMGB1 or depletion of RAGE (receptor for advanced glycation endproducts)-a cell surface receptor for HMGB1. The effect of HMGB1 overexpression on LDL transcytosis was prevented by knockdown of SREBP2. Loss of HMGB1 caused a reduction in the half-life of SREBP2; incubation with LDL caused a significant increase in nuclear localization of HMGB1 that was dependent on SR-BI. Animals lacking endothelial HMGB1 exhibited less acute accumulation of LDL in the aorta 30 minutes after injection and when fed a high-fat diet developed fewer fatty streaks and less atherosclerosis.

Conclusions: Endothelial HMGB1 regulates LDL transcytosis by prolonging the half-life of SREBP2, enhancing SR-BI expression. Translocation of HMGB1 to the nucleus in response to LDL requires SR-BI.
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http://dx.doi.org/10.1161/ATVBAHA.120.314557DOI Listing
January 2021

Staphylococcus aureus Leukocidins Target Endothelial DARC to Cause Lethality in Mice.

Cell Host Microbe 2019 Mar 21;25(3):463-470.e9. Epub 2019 Feb 21.

Department of Microbiology, New York University School of Medicine, New York, NY 10016, USA; Kimmel Center for Biology and Medicine at the Skirball Institute, New York University School of Medicine, New York, NY 10016, USA.

The pathogenesis of Staphylococcus aureus is thought to depend on the production of pore-forming leukocidins that kill leukocytes and lyse erythrocytes. Two leukocidins, Leukocidin ED (LukED) and γ-Hemolysin AB (HlgAB), are necessary and sufficient to kill mice upon infection and toxin challenge. We demonstrate that LukED and HlgAB cause vascular congestion and derangements in vascular fluid distribution that rapidly cause death in mice. The Duffy antigen receptor for chemokines (DARC) on endothelial cells, rather than leukocytes or erythrocytes, is the critical target for lethality. Consistent with this, LukED and HlgAB injure primary human endothelial cells in a DARC-dependent manner, and mice with DARC-deficient endothelial cells are resistant to toxin-mediated lethality. During bloodstream infection in mice, DARC targeting by S. aureus causes increased tissue damage, organ dysfunction, and host death. The potential for S. aureus leukocidins to manipulate vascular integrity highlights the importance of these virulence factors.
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http://dx.doi.org/10.1016/j.chom.2019.01.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468323PMC
March 2019

CD36 mediates albumin transcytosis by dermal but not lung microvascular endothelial cells: role in fatty acid delivery.

Am J Physiol Lung Cell Mol Physiol 2019 05 31;316(5):L740-L750. Epub 2019 Jan 31.

Institute of Medical Science, University of Toronto, Toronto, Canada.

In healthy blood vessels, albumin crosses the endothelium to leave the circulation by transcytosis. However, little is known about the regulation of albumin transcytosis or how it differs in different tissues; its physiological purpose is also unclear. Using total internal reflection fluorescence microscopy, we quantified transcytosis of albumin across primary human microvascular endothelial cells from both lung and skin. We then validated our in vitro findings using a tissue-specific knockout mouse model. We observed that albumin transcytosis was saturable in the skin but not the lung microvascular endothelial cells, implicating a receptor-mediated process. We identified the scavenger receptor CD36 as being both necessary and sufficient for albumin transcytosis across dermal microvascular endothelium, in contrast to the lung where macropinocytosis dominated. Mutations in the apical helical bundle of CD36 prevented albumin internalization by cells. Mice deficient in CD36 specifically in endothelial cells exhibited lower basal permeability to albumin and less basal tissue edema in the skin but not in the lung. Finally, these mice also exhibited a smaller subcutaneous fat layer despite having identical total body weights and circulating fatty acid levels as wild-type animals. In conclusion, CD36 mediates albumin transcytosis in the skin but not the lung. Albumin transcytosis may serve to regulate fatty acid delivery from the circulation to tissues.
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http://dx.doi.org/10.1152/ajplung.00127.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6589589PMC
May 2019

Effect of Tetrodotoxin Pellets in a Rat Model of Postherpetic Neuralgia.

Mar Drugs 2018 Jun 5;16(6). Epub 2018 Jun 5.

Department of Materials Science and Engineering, College of Materials, Xiamen University, Xiamen 361005, China.

Postherpetic neuralgia (PHN) is nerve pain caused by a reactivation of the varicella zoster virus. Medications are used to reduce PHN but their use is limited by serious side effects. Tetrodotoxin (TTX) is a latent neurotoxin that can block neuropathic pain, but its therapeutic index is only 3⁻5 times with intravenous or intramuscular injection. Therefore, we prepared oral TTX pellets and examined their effect in a rat model of PHN induced by resiniferatoxin (RTX). Oral TTX pellets were significantly effective at preventing RTX-induced mechanical and thermal allodynia, and similar to pregabalin. Moreover, oral administration of TTX pellets dose-dependently inhibited RTX-induced PHN compared with intramuscular administration of TTX injection. We also studied the pharmacokinetic profile of TTX pellets. Our results showed that the blood concentration of TTX reached a maximum plasma concentration (C) at around 2 h, with an elimination half-life time (t) of 3.23 ± 1.74 h after intragastric administration. The median lethal dose (LD) of TTX pellets was 517.43 μg/kg via oral administration to rats, while the median effective dose (ED50) was approximately 5.85 μg/kg, and the therapeutic index was 88.45. Altogether, this has indicated that oral TTX pellets greatly enhance safety when compared with TTX injection.
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http://dx.doi.org/10.3390/md16060195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6025269PMC
June 2018

Selenium nanoparticles-loaded chitosan/citrate complex and its protection against oxidative stress in D-galactose-induced aging mice.

J Nanobiotechnology 2017 Dec 20;15(1):92. Epub 2017 Dec 20.

Third Institute of Oceanography, State Oceanic Administration, Xiamen, 361005, People's Republic of China.

Background: Selenium (Se) is an indispensable trace element required for animals and humans, and extra Se-supplement is necessary, especially for those having Se deficiency. Recently, selenium nanoparticles (SeNPs), as a special form of Se supplement, have attracted worldwide attention due to their distinguished properties and excellent bioactivities. In this present study, an eco-friendly and economic way to prepare stable SeNPs was introduced. SeNPs were synthesized in the presence of chitosan (CTS) and then embedded into chitosan/citrate gel, generating selenium nanoparticles-loaded chitosan/citrate complex (SeNPs-C/C). Additionally, the clinical potential of SeNPs-C/C was evaluated by using D-galactose (D-gal)-induced aging mice model.

Results: SeNPs in high uniform with an average diameter of around 50 nm were synthesized in the presence of chitosan, and reversible ionic gelation between chitosan and citrate was utilized to load SeNPs. Subsphaeroidal SeNPs-C/C microspheres of 1-30 μm were obtained by spay-drying. Single SeNPs were physically separated and embedded inside SeNPs-C/C microparticles, with excellent stability and acceptable release. Acute fetal test showed SeNPs-C/C was safer than selenite, with a median lethal dose (LD) of approximately 4-fold to 11-fold of that of selenite. Oral administration of SeNPs-C/C remarkably retarded the oxidative stress of D-gal in Kunming mice by enhancing the activity of antioxidase, as evidenced by its significant protection of the growth, liver, Se retention and antioxidant bio-markers of mice against D-gal.

Conclusions: The design of SeNPs-C/C opens a new path for oral delivery of SeNPs with excellent stability, energy-conservation and environment-friendliness. SeNPs-C/C, as a novel supplement of Se, could be further developed to defend the aging process induced by D-gal.
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http://dx.doi.org/10.1186/s12951-017-0324-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738782PMC
December 2017

SR-BI Mediated Transcytosis of HDL in Brain Microvascular Endothelial Cells Is Independent of Caveolin, Clathrin, and PDZK1.

Front Physiol 2017 30;8:841. Epub 2017 Oct 30.

Department of Biochemistry, University of Toronto, Toronto, ON, Canada.

The vascular endothelium supplying the brain exhibits very low paracellular and transcellular permeability and is a major constituent of the blood-brain barrier. High-density lipoprotein (HDL) crosses the blood-brain barrier by transcytosis, but technical limitations have made it difficult to elucidate its regulation. Using a combination of spinning-disc confocal and total internal reflection fluorescence microscopy, we examined the uptake and transcytosis of HDL by human primary brain microvascular endothelial cell monolayers. Using these approaches, we report that HDL internalization requires dynamin but not clathrin heavy chain and that its internalization and transcytosis are saturable. Internalized HDL partially co-localized with the scavenger receptor BI (SR-BI) and knockdown of SR-BI significantly attenuated HDL internalization. However, we observed that the adaptor protein PDZK1-which is critical to HDL-SR-BI signaling in other tissues-is not required for HDL uptake in these cells. Additionally, while these cells express caveolin, the abundance of caveolae in this tissue is negligible and we find that SR-BI and caveolin do not co-fractionate. Furthermore, direct silencing of caveolin-1 had no impact on the uptake of HDL. Finally, inhibition of endothelial nitric oxide synthase increased HDL internalization while increasing nitric oxide levels had no impact. Together, these data indicate that SR-BI-mediated transcytosis in brain microvascular endothelial cells is distinct from uptake and signaling pathways described for this receptor in other cell types.
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http://dx.doi.org/10.3389/fphys.2017.00841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5670330PMC
October 2017

Influenza Virus Infection Induces Platelet-Endothelial Adhesion Which Contributes to Lung Injury.

J Virol 2016 02 4;90(4):1812-23. Epub 2015 Dec 4.

Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Canada Institute of Medical Science, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Division of Respirology and Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto, Canada

Lung injury after influenza infection is characterized by increased permeability of the lung microvasculature, culminating in acute respiratory failure. Platelets interact with activated endothelial cells and have been implicated in the pathogenesis of some forms of acute lung injury. Autopsy studies have revealed pulmonary microthrombi after influenza infection, and epidemiological studies suggest that influenza vaccination is protective against pulmonary thromboembolism; however, the effect of influenza infection on platelet-endothelial interactions is unclear. We demonstrate that endothelial infection with both laboratory and clinical strains of influenza virus increased the adhesion of human platelets to primary human lung microvascular endothelial cells. Platelets adhered to infected cells as well as to neighboring cells, suggesting a paracrine effect. Influenza infection caused the upregulation of von Willebrand factor and ICAM-1, but blocking these receptors did not prevent platelet-endothelial adhesion. Instead, platelet adhesion was inhibited by both RGDS peptide and a blocking antibody to platelet integrin α5β1, implicating endothelial fibronectin. Concordantly, lung histology from infected mice revealed viral dose-dependent colocalization of viral nucleoprotein and the endothelial marker PECAM-1, while platelet adhesion and fibronectin deposition also were observed in the lungs of influenza-infected mice. Inhibition of platelets using acetylsalicylic acid significantly improved survival, a finding confirmed using a second antiplatelet agent. Thus, influenza infection induces platelet-lung endothelial adhesion via fibronectin, contributing to mortality from acute lung injury. The inhibition of platelets may constitute a practical adjunctive strategy to the treatment of severe infections with influenza.IMPORTANCE There is growing appreciation of the involvement of the lung endothelium in the pathogenesis of severe infections with influenza virus. We have recently shown that the virus can infect human lung endothelial cells, but the functional consequences of this infection are unknown (S. M. Armstrong, C. Wang, J. Tigdi, X. Si, C. Dumpit, S. Charles, A. Gamage, T. J. Moraes, and W. L. Lee, PLoS One 7:e47323, 2012, http://dx.doi.org/10.1371/journal.pone.0047323). Here, we show that this infection causes platelets to adhere to the lung endothelium. Importantly, blocking platelets using two distinct antiplatelet drugs improved survival in a mouse model of severe influenza infection. Thus, platelet inhibition may constitute a novel therapeutic strategy to improve the host response to severe infections with influenza.
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http://dx.doi.org/10.1128/JVI.02599-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4733979PMC
February 2016

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

Cardiovasc Res 2015 Nov 2;108(2):268-77. Epub 2015 Sep 2.

Keenan Research Centre, St Michael's Hospital, 30 Bond Street, Toronto, ON, Canada, M5B 1W8 Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Interdepartmental Division of Critical Care Medicine and the Department of Medicine, University of Toronto, Toronto, ON, Canada

Aims: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved.

Methods And Results: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates.

Conclusion: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.
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http://dx.doi.org/10.1093/cvr/cvv218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614686PMC
November 2015

The Tie2-agonist Vasculotide rescues mice from influenza virus infection.

Sci Rep 2015 Jun 5;5:11030. Epub 2015 Jun 5.

1] Keenan Research Centre for Biomedical Science, St. Michael's Hospital, Toronto, ON [2] Department of Laboratory Medicine and Pathobiology, University of Toronto [3] Interdepartmental Division of Critical Care and Department of Medicine, University of Toronto.

Seasonal influenza virus infections cause hundreds of thousands of deaths annually while viral mutation raises the threat of a novel pandemic strain. Antiviral drugs exhibit limited efficacy unless administered early and may induce viral resistance. Thus, targeting the host response directly has been proposed as a novel therapeutic strategy with the added potential benefit of not eliciting viral resistance. Severe influenza virus infections are complicated by respiratory failure due to the development of lung microvascular leak and acute lung injury. We hypothesized that enhancing lung endothelial barrier integrity could improve the outcome. Here we demonstrate that the Tie2-agonist tetrameric peptide Vasculotide improves survival in murine models of severe influenza, even if administered as late as 72 hours after infection; the benefit was observed using three strains of the virus and two strains of mice. The effect required Tie2, was independent of viral replication and did not impair lung neutrophil recruitment. Administration of the drug decreased lung edema, arterial hypoxemia and lung endothelial apoptosis; importantly, Vasculotide is inexpensive to produce, is chemically stable and is unrelated to any Tie2 ligands. Thus, Vasculotide may represent a novel and practical therapy for severe infections with influenza.
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http://dx.doi.org/10.1038/srep11030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457136PMC
June 2015

Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

Am J Physiol Endocrinol Metab 2015 Jul 5;309(1):E35-44. Epub 2015 May 5.

Cell Biology Program, the Hospital for Sick Children, Toronto, Ontario, Canada;

Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue infiltration by immune cells.
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http://dx.doi.org/10.1152/ajpendo.00611.2014DOI Listing
July 2015

Influenza-Induced Priming and Leak of Human Lung Microvascular Endothelium upon Exposure to Staphylococcus aureus.

Am J Respir Cell Mol Biol 2015 Oct;53(4):459-70

1 Keenan Research Centre for Biomedical Science at St. Michael's Hospital, Toronto, Ontario, Canada.

A major cause of death after influenza virus infection is lung injury due to a bacterial superinfection, yet the mechanism is unknown. Death has been attributed to virus-induced immunosuppression and bacterial overgrowth, but this hypothesis is based on data from the preantibiotic era and animal models that omit antimicrobial therapy. Because of diagnostic uncertainty, most patients with influenza receive antibiotics, making bacterial overgrowth unlikely. Respiratory failure after superinfection presents as acute respiratory distress syndrome, a disorder characterized by lung microvascular leak and edema. The objective of this study was to determine whether the influenza virus sensitizes the lung endothelium to leak upon exposure to circulating bacterial-derived molecular patterns from Staphylococcus aureus. In vitro as well as in vivo models of influenza followed by S. aureus superinfection were used. Molecular mechanisms were explored using molecular biology, knockout mice, and human autopsy specimens. Influenza virus infection sensitized human lung endothelium to leak when challenged with S. aureus, even at low doses of influenza and even when the pathogens were given days apart. Influenza virus increased endothelial expression of TNFR1 both in vitro and in intact lungs, a finding corroborated by human autopsy specimens of patients with influenza. Leak was recapitulated with protein A, a TNFR1 ligand, and sequential infection caused protein A-dependent loss of IκB, cleavage of caspases 8 and 3, and lung endothelial apoptosis. Mice infected sequentially with influenza virus and S. aureus developed significantly increased lung edema that was protein A and TNFR1 dependent. Influenza virus primes the lung endothelium to leak, predisposing patients to acute respiratory distress syndrome upon exposure to S. aureus.
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http://dx.doi.org/10.1165/rcmb.2014-0373OCDOI Listing
October 2015

Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

Mol Biol Cell 2015 Feb 24;26(4):740-50. Epub 2014 Dec 24.

Institute of Medical Sciences, University of Toronto, Toronto, ON M5S 1A8, Canada Keenan Research Centre, St. Michael's Hospital, Toronto, ON M5B 1W8, Canada Department of Microbiology and Immunology, University of Western Ontario, London, ON N6A 5C1, Canada Interdepartmental Division of Critical Care, Department of Medicine, University of Toronto, Toronto, ON M5S 1A8, Canada

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent.
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http://dx.doi.org/10.1091/mbc.E14-08-1307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4325843PMC
February 2015

Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury.

J Am Soc Nephrol 2013 Jul 13;24(8):1274-87. Epub 2013 Jun 13.

Division of Nephrology, Hospital for Sick Children, Toronto, Ontario, Canada.

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.
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http://dx.doi.org/10.1681/ASN.2012090890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3736708PMC
July 2013

Influenza infects lung microvascular endothelium leading to microvascular leak: role of apoptosis and claudin-5.

PLoS One 2012 24;7(10):e47323. Epub 2012 Oct 24.

Institute of Medical Science, University of Toronto, Toronto, Canada.

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047323PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480371PMC
April 2013

Co-regulation of transcellular and paracellular leak across microvascular endothelium by dynamin and Rac.

Am J Pathol 2012 Mar 25;180(3):1308-1323. Epub 2011 Dec 25.

Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; Keenan Research Centre of the Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada; Division of Respirology and Interdepartmental Division of Critical Care Medicine, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada. Electronic address:

Increased permeability of the microvascular endothelium to fluids and proteins is the hallmark of inflammatory conditions such as sepsis. Leakage can occur between (paracellular) or through (transcytosis) endothelial cells, yet little is known about whether these pathways are linked. Understanding the regulation of microvascular permeability is essential for the identification of novel therapies to combat inflammation. We investigated whether transcytosis and paracellular leakage are co-regulated. Using molecular and pharmacologic approaches, we inhibited transcytosis of albumin in primary human microvascular endothelium and measured paracellular permeability. Blockade of transcytosis induced a rapid increase in paracellular leakage that was not explained by decreases in caveolin-1 or increases in activity of nitric oxide synthase. The effect required caveolin-1 but was observed in cells depleted of clathrin, indicating that it was not due to the general inhibition of endocytosis. Inhibiting transcytosis by dynamin blockade increased paracellular leakage concomitantly with the loss of cortical actin from the plasma membrane and the displacement of active Rac from the plasmalemma. Importantly, inhibition of paracellular leakage by sphingosine-1-phosphate, which activates Rac and induces cortical actin, caused a significant increase in transcytosis of albumin in vitro and in an ex vivo whole-lung model. In addition, dominant-negative Rac significantly diminished albumin uptake by endothelia. Our findings indicate that transcytosis and paracellular permeability are co-regulated through a signaling pathway linking dynamin, Rac, and actin.
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http://dx.doi.org/10.1016/j.ajpath.2011.12.002DOI Listing
March 2012

Calcineurin-independent regulation of plasma membrane Ca2+ ATPase-4 in the vascular smooth muscle cell cycle.

Am J Physiol Cell Physiol 2003 Jul 26;285(1):C88-95. Epub 2003 Mar 26.

Department of Medicine, University of Toronto, Toronto, Ontario, Canada M5G-2C4.

Calcineurin mediates repression of plasma membrane Ca2+-ATPase-4 (PMCA4) expression in neurons, whereas c-Myb is known to repress PMCA1 expression in vascular smooth muscle cells (VSMC). Here, we describe a novel mouse VSMC line (MOVAS) in which 45Ca efflux rates decreased 50%, fura 2-AM-based intracellular Ca2+ concentrations ([Ca2+]i) increased twofold, and real-time RT-PCR and Western blot revealed a approximately 40% decrease in PMCA4 expression levels from G0 to G1/S in the cell cycle, where PMCA4 constituted approximately 20% of total PMCA protein. Although calcineurin activity increased fivefold as MOVAS progressed from G0 to G1/S, inhibition of this increase with either BAPTA or retroviral transduction with peptide inhibitors of calcineurin (CAIN), or its downstream target nuclear factor of activated T cells (NFAT) (VIVIT), had no effect on the repression of PMCA4 mRNA expression at G1/S. By contrast, Ca2+-independent activity of the calmodulin-dependent protein kinase-II (CaMK-II) increased eightfold as MOVAS progressed from G0 to G1/S, and treatment with an inhibitor of CaMK-II (KN-93) or transduction of a c-Myb-neutralizing antibody significantly alleviated the G1/S-associated repression of PMCA4. These data show that G1/S-specific PMCA4 repression in proliferating VSMC is brought about by c-Myb and CaMK-II and that calcineurin may regulate cell cycle-associated [Ca2+]i through alternate targets.
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http://dx.doi.org/10.1152/ajpcell.00518.2002DOI Listing
July 2003

Nucleotide triphosphatase activity of the N-terminal nucleotide-binding domains of the multidrug resistance proteins P-glycoprotein and MRP1.

Biochem Biophys Res Commun 2002 Aug;296(2):388-94

Membrane Protein Laboratory, Department of Physiology and Biophysics, The University of Texas Medical Branch Galveston, 301 University Boulevard, Galveston, TX 77555-0437, USA.

The multidrug resistance proteins P-glycoprotein (Pgp) and MRP1 are drug-efflux pumps. In this study, we compared the nucleotide triphosphatase activities of the isolated N-terminal nucleotide binding domains (NBD1) of Pgp and MRP1, and explored the potential role of the phosphorylation target domain of Pgp on the regulation of Pgp NBD1 ATPase activity. We found that: (1) the NBD1s of Pgp and MRP1 have ATPase and GTPase activities, (2) the K(m)s of Pgp NBD1 for ATP and GTP hydrolysis are identical, while the K(m) of MRP1 NBD1 for ATP is lower than that for GTP, and (3) phosphorylation of MLD by PKA or PKC produces a marginal increase of V(max) for ATP hydrolysis, without affecting the affinity for ATP. These results show efficient GTP hydrolysis by the NBD1s of Pgp and MRP1, and a minor role of phosphorylation in the control of Pgp NBD1 ATPase activity.
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http://dx.doi.org/10.1016/s0006-291x(02)00878-1DOI Listing
August 2002
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