Publications by authors named "Warren L Lee"

62 Publications

Interactions of Influenza and SARS-CoV-2 with the Lung Endothelium: Similarities, Differences, and Implications for Therapy.

Viruses 2021 01 22;13(2). Epub 2021 Jan 22.

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada.

Respiratory viruses such as influenza and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are a constant threat to public health given their ability to cause global pandemics. Infection with either virus may lead to aberrant host responses, such as excessive immune cell recruitment and activation, dysregulated inflammation, and coagulopathy. These may contribute to the development of lung edema and respiratory failure. An increasing amount of evidence suggests that lung endothelial cells play a critical role in the pathogenesis of both viruses. In this review, we discuss how infection with influenza or SARS-CoV-2 may induce endothelial dysfunction. We compare the effects of infection of these two viruses, how they may contribute to pathogenesis, and discuss the implications for potential treatment. Understanding the differences between the effects of these two viruses on lung endothelial cells will provide important insight to guide the development of therapeutics.
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http://dx.doi.org/10.3390/v13020161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911974PMC
January 2021

Radiation Impacts Early Atherosclerosis by Suppressing Intimal LDL Accumulation.

Circ Res 2021 Feb 5;128(4):530-543. Epub 2021 Jan 5.

Toronto General Hospital Research Institute (J.I., C.A.S., S.J.H., M.G.A., J.A., S.A.D., T.E., J.M., N.I., H.M.I., C.K.P., X.G., K.T., J.J.-B., S.E., C.S.R., S.A.M., M.I.C.), University Health Network, Toronto, Canada.

Rationale: Bone marrow transplantation (BMT) is used frequently to study the role of hematopoietic cells in atherosclerosis, but aortic arch lesions are smaller in mice after BMT.

Objective: To identify the earliest stage of atherosclerosis inhibited by BMT and elucidate potential mechanisms.

Methods And Results: mice underwent total body γ-irradiation, bone marrow reconstitution, and 6-week recovery. Atherosclerosis was studied in the ascending aortic arch and compared with mice without BMT. In BMT mice, neutral lipid and myeloid cell topography were lower in lesions after feeding a cholesterol-rich diet for 3, 6, and 12 weeks. Lesion coalescence and height were suppressed dramatically in mice post-BMT, whereas lateral growth was inhibited minimally. Targeted radiation to the upper thorax alone reproduced the BMT phenotype. Classical monocyte recruitment, intimal myeloid cell proliferation, and apoptosis did not account for the post-BMT phenotype. Neutral lipid accumulation was reduced in 5-day lesions, thus we developed quantitative assays for LDL (low-density lipoprotein) accumulation and paracellular leakage using DiI-labeled human LDL and rhodamine B-labeled 70 kD dextran. LDL accumulation was dramatically higher in the intima of relative to mice, and was inhibited by injection of HDL mimics, suggesting a regulated process. LDL, but not dextran, accumulation was lower in mice post-BMT both at baseline and in 5-day lesions. Since the transcript abundance of molecules implicated in LDL transcytosis was not significantly different in the post-BMT intima, transcriptomics from whole aortic arch intima, and at single-cell resolution, was performed to give insights into pathways modulated by BMT.

Conclusions: Radiation exposure inhibits LDL entry into the aortic intima at baseline and the earliest stages of atherosclerosis. Single-cell transcriptomic analysis suggests that LDL uptake by endothelial cells is diverted to lysosomal degradation and reverse cholesterol transport pathways. This reduces intimal accumulation of lipid and impacts lesion initiation and growth.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.316539DOI Listing
February 2021

BMP-9 and LDL crosstalk regulates ALK-1 endocytosis and LDL transcytosis in endothelial cells.

J Biol Chem 2020 12 23;295(52):18179-18188. Epub 2020 Oct 23.

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA; Vascular Biology and Therapeutics Program, Yale University School of Medicine, New Haven, Connecticut, USA. Electronic address:

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9-mediated internalization of ALK-1, BMP-9-dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9-induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9-mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.
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http://dx.doi.org/10.1074/jbc.RA120.015680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7939458PMC
December 2020

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

Transendothelial transport of lipoproteins.

Atherosclerosis 2020 12 25;315:111-125. Epub 2020 Sep 25.

Keenan Centre for Biomedical Research, St. Michael's Hospital, Toronto, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Canada; Interdepartmental Division of Critical Care, Department of Medicine, University of Toronto, Canada; Department of Biochemistry, University of Toronto, Canada; Institute of Medical Science, University of Toronto, Canada. Electronic address:

The accumulation of low-density lipoproteins (LDL) in the arterial wall plays a pivotal role in the initiation and pathogenesis of atherosclerosis. Conversely, the removal of cholesterol from the intima by cholesterol efflux to high density lipoproteins (HDL) and subsequent reverse cholesterol transport shall confer protection against atherosclerosis. To reach the subendothelial space, both LDL and HDL must cross the intact endothelium. Traditionally, this transit is explained by passive filtration. This dogma has been challenged by the identification of several rate-limiting factors namely scavenger receptor SR-BI, activin like kinase 1, and caveolin-1 for LDL as well as SR-BI, ATP binding cassette transporter G1, and endothelial lipase for HDL. In addition, estradiol, vascular endothelial growth factor, interleukins 6 and 17, purinergic signals, and sphingosine-1-phosphate were found to regulate transendothelial transport of either LDL or HDL. Thorough understanding of transendothelial lipoprotein transport is expected to elucidate new therapeutic targets for the treatment or prevention of atherosclerotic cardiovascular disease and the development of strategies for the local delivery of drugs or diagnostic tracers into diseased tissues including atherosclerotic lesions.
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http://dx.doi.org/10.1016/j.atherosclerosis.2020.09.020DOI Listing
December 2020

Photoacoustic imaging of kidney fibrosis for assessing pretransplant organ quality.

JCI Insight 2020 05 21;5(10). Epub 2020 May 21.

Institute for Biomedical Engineering, Science and Technology (iBEST), a partnership between Ryerson University and St. Michael's Hospital, Toronto, Canada.

Roughly 10% of the world's population has chronic kidney disease (CKD). In its advanced stages, CKD greatly increases the risk of hospitalization and death. Although kidney transplantation has revolutionized the care of advanced CKD, clinicians have limited ways of assessing donor kidney quality. Thus, optimal donor kidney-recipient matching cannot be performed, meaning that some patients receive damaged kidneys that function poorly. Fibrosis is a form of chronic damage often present in donor kidneys, and it is an important predictor of future renal function. Currently, no safe, easy-to-perform technique exists that accurately quantifies renal fibrosis. We describe a potentially novel photoacoustic (PA) imaging technique that directly images collagen, the principal component of fibrotic tissue. PA imaging noninvasively quantifies whole kidney fibrotic burden in mice, and cortical fibrosis in pig and human kidneys, with outstanding accuracy and speed. Remarkably, 3-dimensional PA imaging exhibited sufficiently high resolution to capture intrarenal variations in collagen content. We further show that PA imaging can be performed in a setting that mimics human kidney transplantation, suggesting the potential for rapid clinical translation. Taken together, our data suggest that PA collagen imaging is a major advance in fibrosis quantification that could have widespread preclinical and clinical impact.
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http://dx.doi.org/10.1172/jci.insight.136995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259526PMC
May 2020

The Endothelial Barrier Is not Rate-limiting to Insulin Action in the Myocardium of Male Mice.

Endocrinology 2020 04;161(4)

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

To act on tissues, circulating insulin must perfuse the relevant organ and then leave the bloodstream by crossing the endothelium-a process known as insulin delivery. It has been postulated that the continuous endothelium is a rate-limiting barrier to insulin delivery but existing data are contradictory. This conflict is in part due to the limitations of current models, including the inability to maintain a constant blood pressure in animals and the absence of shear stress in cultured cells. We developed a murine cardiac ex vivo perfusion model that delivers insulin to the heart in situ at a constant flow. We hypothesized that if the endothelial barrier were rate-limiting to insulin delivery, increasing endothelial permeability would accelerate insulin action. The kinetics of myocardial insulin action were determined in the presence or absence of agents that increased endothelial permeability. Permeability was measured using Evans Blue, which binds with high affinity to albumin. During our experiments, the myocardium remained sensitive to insulin and the vasculature retained barrier integrity. Perfusion with insulin induced Akt phosphorylation in myocytes but not in the endothelium. Infusion of platelet-activating factor or vascular endothelial growth factor significantly increased permeability to albumin without altering insulin action. Amiloride, an inhibitor of fluid-phase uptake, also did not alter insulin action. These data suggest that the endothelial barrier is not rate limiting to insulin's action in the heart; its passage out of the coronary circulation is consistent with diffusion or convection. Modulation of transendothelial transport to overcome insulin resistance is unlikely to be a viable therapeutic strategy.
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http://dx.doi.org/10.1210/endocr/bqaa029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7069687PMC
April 2020

Long noncoding RNA integrates a DNA-PK-mediated DNA damage response and vascular senescence.

Sci Transl Med 2020 02;12(531)

Department of Medicine, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Long noncoding RNAs (lncRNAs) are emerging regulators of biological processes in the vessel wall; however, their role in atherosclerosis remains poorly defined. We used RNA sequencing to profile lncRNAs derived specifically from the aortic intima of mice on a high-cholesterol diet during lesion progression and regression phases. We found that the evolutionarily conserved lncRNA small nucleolar host gene-12 () is highly expressed in the vascular endothelium and decreases during lesion progression. knockdown accelerated atherosclerotic lesion formation by 2.4-fold in mice by increased DNA damage and senescence in the vascular endothelium, independent of effects on lipid profile or vessel wall inflammation. Conversely, intravenous delivery of protected the tunica intima from DNA damage and atherosclerosis. LncRNA pulldown in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that interacted with DNA-dependent protein kinase (DNA-PK), an important regulator of the DNA damage response. The absence of reduced the DNA-PK interaction with its binding partners Ku70 and Ku80, abrogating DNA damage repair. Moreover, the anti-DNA damage agent nicotinamide riboside (NR), a clinical-grade small-molecule activator of NAD, fully rescued the increases in lesional DNA damage, senescence, and atherosclerosis mediated by knockdown. expression was also reduced in pig and human atherosclerotic specimens and correlated inversely with DNA damage and senescent markers. These findings reveal a role for this lncRNA in regulating DNA damage repair in the vessel wall and may have implications for chronic vascular disease states and aging.
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http://dx.doi.org/10.1126/scitranslmed.aaw1868DOI Listing
February 2020

Reply to Mehmood: Adrenomedullin: A Double-edged Sword in Septic Shock and Heart Failure Therapeutics?

Authors:
Warren L Lee

Am J Respir Crit Care Med 2020 05;201(9):1165

Unity Health TorontoToronto, Ontario, CanadaandUniversity of TorontoToronto, Ontario, Canada.

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http://dx.doi.org/10.1164/rccm.202001-0072LEDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193863PMC
May 2020

Inflammation without Vascular Leakage. Science Fiction No Longer?

Am J Respir Crit Care Med 2019 12;200(12):1472-1476

Department of Critical Care Medicine and.

Vascular leakage is a characteristic of critical illnesses such as septic shock and acute respiratory distress syndrome. It results in hypotension and tissue edema and contributes to organ dysfunction. It has long been taught that increased vascular permeability is a natural consequence of inflammation; in particular, many clinicians believe that it occurs inevitably during leukocyte recruitment to a site of infection. In fact, abundant research now indicates that vascular leakage and leukocyte emigration do not necessarily occur together in a blood vessel. The molecular mechanisms underpinning these processes-allowing leukocytes to exit the circulation without increasing vascular permeability-are starting to be elucidated and establish vascular leakage as a viable therapeutic target. Several preclinical studies indicate that vascular leakage can be reduced without impairing cytokine production, leukocyte recruitment, and pathogen clearance. The realization that leukocyte traffic and vascular permeability can be regulated separately should spur development of therapies that decrease vascular leakage and tissue edema without compromising the immune response.
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http://dx.doi.org/10.1164/rccm.201905-1011CPDOI Listing
December 2019

Caveolin-1 Regulates Atherogenesis by Attenuating Low-Density Lipoprotein Transcytosis and Vascular Inflammation Independently of Endothelial Nitric Oxide Synthase Activation.

Circulation 2019 07 3;140(3):225-239. Epub 2019 Jun 3.

Vascular Biology and Therapeutics Program (C.M.R., X.Z., N.R., B.A., J.R.K., V.U., Y.S., W.C.S., C.F.-H.), Yale University School of Medicine, New Haven, CT.

Background: Atherosclerosis is driven by synergistic interactions between pathological, biomechanical, inflammatory, and lipid metabolic factors. Our previous studies demonstrated that absence of caveolin-1 (Cav1)/caveolae in hyperlipidemic mice strongly inhibits atherosclerosis, which was attributed to activation of endothelial nitric oxide (NO) synthase (eNOS) and increased production of NO and reduced inflammation and low-density lipoprotein trafficking. However, the contribution of eNOS activation and NO production in the athero-protection of Cav1 and the exact mechanisms by which Cav1/caveolae control the pathogenesis of diet-induced atherosclerosis are still not clear.

Methods: Triple-knockout mouse lacking expression of eNOS, Cav1, and Ldlr were generated to explore the role of NO production in Cav1-dependent athero-protective function. The effects of Cav1 on lipid trafficking, extracellular matrix remodeling, and vascular inflammation were studied both in vitro and in vivo with a mouse model of diet-induced atherosclerosis. The expression of Cav1 and distribution of caveolae regulated by flow were analyzed by immunofluorescence staining and transmission electron microscopy.

Results: We found that absence of Cav1 significantly suppressed atherogenesis in LdlreNOS mice, demonstrating that athero-suppression is independent of increased NO production. Instead, we find that the absence of Cav1/caveolae inhibited low-density lipoprotein transport across the endothelium and proatherogenic fibronectin deposition and disturbed flow-mediated endothelial cell inflammation. Consistent with the idea that Cav1/caveolae may play a role in early flow-dependent inflammatory priming, distinct patterns of Cav1 expression and caveolae distribution were observed in athero-prone and athero-resistant areas of the aortic arch even in wild-type mice.

Conclusions: These findings support a role for Cav1/caveolae as a central regulator of atherosclerosis that links biomechanical, metabolic, and inflammatory pathways independently of endothelial eNOS activation and NO production.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.038571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6778687PMC
July 2019

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

Estrogen Inhibits LDL (Low-Density Lipoprotein) Transcytosis by Human Coronary Artery Endothelial Cells via GPER (G-Protein-Coupled Estrogen Receptor) and SR-BI (Scavenger Receptor Class B Type 1).

Arterioscler Thromb Vasc Biol 2018 10;38(10):2283-2294

From the Keenan Centre for Biomedical Research, St. Michael's Hospital, Toronto, Canada (S.G., F.N.N., M.G.S., W.L.L.).

Objective- The atheroprotective effects of estrogen are independent of circulating lipid levels. Whether estrogen regulates transcytosis of LDL (low-density lipoprotein) across the coronary endothelium is unknown. Approach and Results- Using total internal reflection fluorescence microscopy, we quantified transcytosis of LDL across human coronary artery endothelial cells from multiple donors. LDL transcytosis was significantly higher in cells from men compared with premenopausal women. Estrogen significantly attenuated LDL transcytosis by endothelial cells from male but not female donors; transcytosis of albumin was not affected. Estrogen caused downregulation of endothelial SR-BI (scavenger receptor class B type 1), and overexpression of SR-BI was sufficient to restore LDL transcytosis. Similarly, depletion of SR-BI by siRNA attenuated endothelial LDL transcytosis and prevented any further effect of estrogen. In contrast, treatment with estrogen had no effect on SR-BI expression by liver cells. Inhibition of estrogen receptors α and β had no effect on estrogen-mediated attenuation of LDL transcytosis. However, estrogen's effect on LDL transcytosis was blocked by depletion of the GPER (G-protein-coupled estrogen receptor). GPER was found to be enriched in endothelial cells compared with hepatocytes and is reported to signal via transactivation of the EGFR (epidermal growth factor receptor); inhibition of EGFR prevented the effect of estrogen on LDL transcytosis and SR-BI mRNA. Last, SR-BI expression was significantly higher in human coronary artery endothelial cells from male compared with premenopausal female donors. Conclusions- Estrogen significantly inhibits LDL transcytosis by downregulating endothelial SR-BI; this effect requires GPER.
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http://dx.doi.org/10.1161/ATVBAHA.118.310792DOI Listing
October 2018

Immunotherapy for Sepsis: A Good Idea or Another Dead End?

Authors:
Warren L Lee

Anesthesiology 2018 07;129(1):5-7

From the Keenan Research Centre for Biomedical Research, St. Michael's Hospital, Toronto, Canada; and the Interdepartmental Division of Critical Care, Department of Medicine, University of Toronto, Toronto, Canada.

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http://dx.doi.org/10.1097/ALN.0000000000002237DOI Listing
July 2018

Should basic science matter to clinicians?

Lancet 2018 02;391(10119):410-412

Keenan Research Centre for Biomedical Science, St Michael's Hospital, Toronto, ON M5B 1W8, Canada; Interdepartmental Division of Critical Care, University of Toronto, Toronto, ON, Canada; Department of Medicine, University of Toronto, Toronto, ON, Canada. Electronic address:

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http://dx.doi.org/10.1016/S0140-6736(18)30199-5DOI Listing
February 2018

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

Transcellular vesicular transport in epithelial and endothelial cells: Challenges and opportunities.

Traffic 2018 01 21;19(1):5-18. Epub 2017 Nov 21.

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

Vesicle-mediated transcellular transport or simply "transcytosis" is a cellular process used to shuttle macromolecules such as lipoproteins, antibodies, and albumin from one surface of a polarized cell to the other. This mechanism is in contrast to the transit of small molecules such as anions, cations and amino acids that occur via uptake, diffusion through the cytosol and release and is also distinct from paracellular leak between cells. Importantly, transcytosis has evolved as a process to selectively move macromolecules between 2 neighboring yet unique microenvironments within a multicellular organism. Examples include the movement of lipoproteins out of the circulatory system and into tissues and the delivery of immunoglobulins to mucosal surfaces. Regardless of whether the transport is conducted by endothelial or epithelial cells, the process often involves receptor-mediated uptake of a ligand into an endocytic vesicle, regulated transit of the carrier through the cytoplasm and release of the cargo via an exocytic event. While transcytosis has been examined in detail in epithelial cells, for both historical and technical reasons, the process is less understood in endothelial cells. Here, we spotlight aspects of epithelial transcytosis including recent findings and review the comparative dearth of knowledge regarding the process in endothelial cells highlighting the opportunity for further study.
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http://dx.doi.org/10.1111/tra.12533DOI Listing
January 2018

Development of a zebrafish sepsis model for high-throughput drug discovery.

Mol Med 2017 07 7;23:134-148. Epub 2017 Jun 7.

Zebrafish Centre for Advanced Drug Discovery, St. Michael's Hospital, 209 Victoria St, Toronto, Ontario, Canada M5B 1T8.

Sepsis is a leading cause of death worldwide. Current treatment modalities remain largely supportive. Intervention strategies focused on inhibiting specific mediators of the inflammatory host response have been largely unsuccessful, a consequence of an inadequate understanding of the complexity and heterogeneity of the innate immune response. Moreover, the conventional drug development pipeline is time consuming and expensive and the low success rates associated with cell-based screens underline the need for whole organism screening strategies, especially for complex pathological processes. Here, we established an LPS-induced zebrafish endotoxemia model, which exhibits the major hallmarks of human sepsis including, edema and tissue/organ damage, increased vascular permeability and vascular leakage accompanied by an altered expression of cellular junction proteins, increased cytokine expression, immune cell activation and ROS production, reduced circulation and increased platelet aggregation. We tested the suitability of the model for phenotype-based drug screening using three primary readouts: mortality, vascular leakage, and ROS production. Preliminary screening identified fasudil, a drug known to protect against vascular leakage in murine models, as a lead hit thereby validating the utility of our model for sepsis drug screens. This zebrafish sepsis model has the potential to rapidly analyze sepsis associated pathologies and cellular processes in the whole organism, as well as to screen and validate large numbers of compounds that can modify sepsis pathology in .
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http://dx.doi.org/10.2119/molmed.2016.00188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522968PMC
July 2017

Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells.

Nat Commun 2016 11 21;7:13516. Epub 2016 Nov 21.

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.
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http://dx.doi.org/10.1038/ncomms13516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5121336PMC
November 2016

Research projects in the Surgeon-Scientist and Clinician-Investigator programs at the University of Toronto (1987-2016): a cohort study.

CMAJ Open 2016 Jul-Sep;4(3):E444-E447. Epub 2016 Aug 22.

Departments of Anesthesia (Goldenberg, Steinberg); Surgery (Rutka, Cabral, Kapus); Medicine (Chen, Lee); Pediatrics (Rosenblum), University of Toronto; Keenan Research Centre for Biomedical Science (Kapus, Lee), Li Ka Shing Knowledge Institute at St. Michael's Hospital; Interdepartmental Division of Critical Care Medicine (Lee), University of Toronto, Toronto, Ont.

Background: Physicians have traditionally been at the forefront of medical research, bringing clinical questions to the laboratory and returning with ideas for treatment. However, we have anecdotally observed a decline in the popularity of basic science research among trainees. We hypothesized that fewer resident physicians have been pursuing basic science research training over time.

Methods: We examined records from residents in the Surgeon-Scientist and Clinician-Investigator programs at the University of Toronto (1987-2016). Research by residents was categorized independently by 2 raters as basic science, clinical epidemiology or education-related based on the title of the project, the name of the supervisor and Pubmed searches. The study population was divided into quintiles of time, and the proportion pursuing basic science training in each quintile was calculated.

Results: Agreement between the raters was 100%; the categorization of the research topic remained unclear in 9 cases. The proportion of trainees pursuing basic science training dropped by 60% from 1987 to 2016 ( = 0.005).

Interpretation: Significantly fewer residents in the Surgeon-Scientist and Clinician-Investigator Programs at the University of Toronto are pursuing training in the basic sciences as compared with previous years.
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http://dx.doi.org/10.9778/cmajo.20160062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5047840PMC
August 2016

Endothelial Transcytosis of Insulin: Does It Contribute to Insulin Resistance?

Physiology (Bethesda) 2016 09;31(5):336-45

Cell Biology Program, The Hospital for Sick Children, Toronto, Canada; Department of Biochemistry, University of Toronto, Toronto, Canada; Paediatrics, and Physiology, University of Toronto, Toronto, Canada.

Most research on insulin resistance has focused on impaired signaling at the level of target tissues like skeletal muscle. Insulin delivery is also important and includes recruitment and perfusion of capillaries bearing insulin, but also the transit of insulin across the capillary endothelium. The mechanisms of this second stage (insulin transcytosis) and whether it contributes to insulin resistance remain uncertain.
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http://dx.doi.org/10.1152/physiol.00010.2016DOI Listing
September 2016

Adhesion Molecules: Master Controllers of the Circulatory System.

Compr Physiol 2016 Mar 15;6(2):945-73. Epub 2016 Mar 15.

Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, University of Colorado, Aurora, Colorado, USA.

This manuscript will review our current understanding of cellular adhesion molecules (CAMs) relevant to the circulatory system, their physiological role in control of vascular homeostasis, innate and adaptive immune responses, and their importance in pathophysiological (disease) processes such as acute lung injury, atherosclerosis, and pulmonary hypertension. This is a complex and rapidly changing area of research that is incompletely understood. By design, we will begin with a brief overview of the structure and classification of the major groups of adhesion molecules and their physiological functions including cellular adhesion and signaling. The role of specific CAMs in the process of platelet aggregation and hemostasis and leukocyte adhesion and transendothelial migration will be reviewed as examples of the complex and cooperative interplay between CAMs during physiological and pathophysiological processes. The role of the endothelial glycocalyx and the glycobiology of this complex system related to inflammatory states such as sepsis will be reviewed. We will then focus on the role of adhesion molecules in the pathogenesis of specific disease processes involving the lungs and cardiovascular system. The potential of targeting adhesion molecules in the treatment of immune and inflammatory diseases will be highlighted in the relevant sections throughout the manuscript.
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http://dx.doi.org/10.1002/cphy.c150020DOI Listing
March 2016

Prostaglandin I2 Receptor Agonism Preserves β-Cell Function and Attenuates Albuminuria Through Nephrin-Dependent Mechanisms.

Diabetes 2016 05 11;65(5):1398-409. Epub 2016 Feb 11.

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge Institute of St. Michael's Hospital, Toronto, Ontario, Canada

Discovery of common pathways that mediate both pancreatic β-cell function and end-organ function offers the opportunity to develop therapies that modulate glucose homeostasis and separately slow the development of diabetes complications. Here, we investigated the in vitro and in vivo effects of pharmacological agonism of the prostaglandin I2 (IP) receptor in pancreatic β-cells and in glomerular podocytes. The IP receptor agonist MRE-269 increased intracellular 3',5'-cyclic adenosine monophosphate (cAMP), augmented glucose-stimulated insulin secretion (GSIS), and increased viability in MIN6 β-cells. Its prodrug form, selexipag, augmented GSIS and preserved islet β-cell mass in diabetic mice. Determining that this preservation of β-cell function is mediated through cAMP/protein kinase A (PKA)/nephrin-dependent pathways, we found that PKA inhibition, nephrin knockdown, or targeted mutation of phosphorylated nephrin tyrosine residues 1176 and 1193 abrogated the actions of MRE-269 in MIN6 cells. Because nephrin is important to glomerular permselectivity, we next set out to determine whether IP receptor agonism similarly affects nephrin phosphorylation in podocytes. Expression of the IP receptor in podocytes was confirmed in cultured cells by immunoblotting and quantitative real-time PCR and in mouse kidneys by immunogold electron microscopy, and its agonism 1) increased cAMP, 2) activated PKA, 3) phosphorylated nephrin, and 4) attenuated albumin transcytosis. Finally, treatment of diabetic endothelial nitric oxide synthase knockout mice with selexipag augmented renal nephrin phosphorylation and attenuated albuminuria development independently of glucose change. Collectively, these observations describe a pharmacological strategy that posttranslationally modifies nephrin and the effects of this strategy in the pancreas and in the kidney.
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http://dx.doi.org/10.2337/db15-0783DOI Listing
May 2016

Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

Am J Physiol Lung Cell Mol Physiol 2016 04 5;310(8):L720-32. Epub 2016 Feb 5.

Division of Pulmonary Pharmacology, Research Center Borstel, Borstel, Germany; Institute of Pharmacology and Toxicology, RWTH Aachen, Aachen, Germany

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.
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http://dx.doi.org/10.1152/ajplung.00157.2015DOI Listing
April 2016

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

Is basic science disappearing from medicine? The decline of biomedical research in the medical literature.

FASEB J 2016 Feb 14;30(2):515-8. Epub 2015 Oct 14.

*Department of Anesthesia, Department of Surgery, Interdepartmental Division of Critical Care, and Department of Medicine, University of Toronto, Toronto, Ontario, Canada; and Keenan Research Centre for Biomedical Science, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada

Explosive growth in our understanding of genomics and molecular biology have fueled calls for the pursuit of personalized medicine, the notion of harnessing biologic variability to provide patient-specific care. This vision will necessitate a deep understanding of the underlying pathophysiology in each patient. Medical journals play a pivotal role in the education of trainees and clinicians, yet we suspected that the amount of basic science in the top medical journals has been in decline. We conducted an automated search strategy in PubMed to identify basic science articles and calculated the proportion of articles dealing with basic science in the highest impact journals for 8 different medical specialties from 1994 to 2013. We observed a steep decline (40-60%) in such articles over time in almost all of the journals examined. This rapid decline in basic science from medical journals is likely to affect practitioners' understanding of and interest in the basic mechanisms of disease and therapy. In this Life Sciences Forum, we discuss why this decline may be occurring and what it means for the future of science and medicine.
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http://dx.doi.org/10.1096/fj.15-281758DOI Listing
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

Role of Transient Receptor Potential Vanilloid 4 in Neutrophil Activation and Acute Lung Injury.

Am J Respir Cell Mol Biol 2016 Mar;54(3):370-83

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

The cation channel transient receptor potential vanilloid (TRPV) 4 is expressed in endothelial and immune cells; however, its role in acute lung injury (ALI) is unclear. The functional relevance of TRPV4 was assessed in vivo, in isolated murine lungs, and in isolated neutrophils. Genetic deficiency of TRPV4 attenuated the functional, histological, and inflammatory hallmarks of acid-induced ALI. Similar protection was obtained with prophylactic administration of the TRPV4 inhibitor, GSK2193874; however, therapeutic administration of the TRPV4 inhibitor, HC-067047, after ALI induction had no beneficial effect. In isolated lungs, platelet-activating factor (PAF) increased vascular permeability in lungs perfused with trpv4(+/+) more than with trpv4(-/-) blood, independent of lung genotype, suggesting a contribution of TRPV4 on blood cells to lung vascular barrier failure. In neutrophils, TRPV4 inhibition or deficiency attenuated the PAF-induced increase in intracellular calcium. PAF induced formation of epoxyeicosatrienoic acids by neutrophils, which, in turn, stimulated TRPV4-dependent Ca(2+) signaling, whereas inhibition of epoxyeicosatrienoic acid formation inhibited the Ca(2+) response to PAF. TRPV4 deficiency prevented neutrophil responses to proinflammatory stimuli, including the formation of reactive oxygen species, neutrophil adhesion, and chemotaxis, putatively due to reduced activation of Rac. In chimeric mice, however, the majority of protective effects in acid-induced ALI were attributable to genetic deficiency of TRPV4 in parenchymal tissue, whereas TRPV4 deficiency in circulating blood cells primarily reduced lung myeloperoxidase activity. Our findings identify TRPV4 as novel regulator of neutrophil activation and suggest contributions of both parenchymal and neutrophilic TRPV4 in the pathophysiology of ALI.
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http://dx.doi.org/10.1165/rcmb.2014-0225OCDOI Listing
March 2016
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