Publications by authors named "Myron I Cybulsky"

59 Publications

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

Anti-Inflammatory Actions of Soluble Ninjurin-1 Ameliorate Atherosclerosis.

Circulation 2020 Nov 4;142(18):1736-1751. Epub 2020 Sep 4.

Immune and Vascular Cell Network Research Center, National Creative Initiatives, Department of Life Sciences (S.J., T.K.K., M.-N.L., S.-K.S., S.S., J.J., H.Y.K., S.K., G.T.O.), Ewha Womans University, Seoul, Korea.

Background: Macrophages produce many inflammation-associated molecules, released by matrix metalloproteinases, such as adhesion molecules, and cytokines, as well, which play a crucial role in atherosclerosis. In this context, we investigated the relationship between Ninjurin-1 (Ninj1 [nerve injury-induced protein]), a novel matrix metalloproteinase 9 substrate, expression, and atherosclerosis progression.

Methods: Ninj1 expression and atherosclerosis progression were assessed in atherosclerotic aortic tissue and serum samples from patients with coronary artery disease and healthy controls, and atheroprone apolipoprotein e-deficient () and wild-type mice, as well. mice lacking systemic Ninj1 expression () were generated to assess the functional effects of Ninj1. Bone marrow transplantation was also used to generate low-density lipoprotein receptor-deficient () mice that lack specifically in bone marrow-derived cells. Mice were fed a Western diet for 5 to 23 weeks, and atherosclerotic lesions were investigated. The anti-inflammatory role of Ninj1 was verified by treating macrophages and mice with the peptides Ninj1 (ML56) and Ninj1 (PN12), which mimic the soluble form of Ninj1 (sNinj1).

Results: Our in vivo results conclusively showed a correlation between Ninj1 expression in aortic macrophages and the extent of human and mouse atherosclerotic lesions. -deficient macrophages promoted proinflammatory gene expression by activating mitogen-activated protein kinase and inhibiting the phosphoinositide 3-kinase/Akt signaling pathway. Whole-body and bone marrow-specific deficiencies significantly increased monocyte recruitment and macrophage accumulation in atherosclerotic lesions through elevated macrophage-mediated inflammation. Macrophage Ninj1 was directly cleaved by matrix metalloproteinase 9 to generate a soluble form that exhibited antiatherosclerotic effects, as assessed in vitro and in vivo. Treatment with the sNinj1-mimetic peptides, ML56 and PN12, reduced proinflammatory gene expression in human and mouse classically activated macrophages, thereby attenuating monocyte transendothelial migration. Moreover, continuous administration of mPN12 alleviated atherosclerosis by inhibiting the enhanced monocyte recruitment and inflammation characteristics of this disorder in mice, regardless of the presence of Ninj1.

Conclusions: Ninj1 is a novel matrix metalloproteinase 9 substrate in macrophages, and sNinj1 is a secreted atheroprotective protein that regulates macrophage inflammation and monocyte recruitment in atherosclerosis. Moreover, sNinj1-mediated anti-inflammatory effects are conserved in human macrophages and likely contribute to human atherosclerosis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.120.046907DOI Listing
November 2020

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

Circ Res 2020 Jul 16;127(3):402-426. Epub 2020 Jul 16.

La Jolla Institute for Immunology, CA (C.C.H., Y.G., H.Q.D., K.L.).

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2 foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, , which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.316903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371244PMC
July 2020

Epigenetic Heterogeneity and Mitotic Heritability Prime Endothelial Cell Gene Induction.

J Immunol 2020 03 29;204(5):1173-1187. Epub 2020 Jan 29.

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada;

Homogeneous populations of mature differentiated primary cell types can display variable responsiveness to extracellular stimuli, although little is known about the underlying mechanisms that govern such heterogeneity at the level of gene expression. In this article, we show that morphologically homogenous human endothelial cells exhibit heterogeneous expression of VCAM1 after TNF-α stimulation. Variability in VCAM1 expression was not due to stochasticity of intracellular signal transduction but rather to preexisting established heterogeneous states of promoter DNA methylation that were generationally conserved through mitosis. Variability in DNA methylation of the promoter resulted in graded RelA/p65 and RNA polymerase II binding that gave rise to a distribution of transcription in the population after TNF-α stimulation. Microarray analysis and single-cell RNA sequencing revealed that a number of cytokine-inducible genes shared this heterogeneous response pattern. These results show that heritable epigenetic heterogeneity is fundamental in inflammatory signaling and highlight as a metastable epiallele.
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http://dx.doi.org/10.4049/jimmunol.1900744DOI Listing
March 2020

c-Myb Exacerbates Atherosclerosis through Regulation of Protective IgM-Producing Antibody-Secreting Cells.

Cell Rep 2019 05;27(8):2304-2312.e6

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S1A1, Canada; Department of Immunology, University of Toronto, Toronto, ON M5S1A1, Canada; Toronto General Research Institute, University Health Network, Toronto, ON M5G1L7, Canada; Peter Munk Cardiac Centre, Toronto, ON M5G1L7, Canada. Electronic address:

Mechanisms that govern transcriptional regulation of inflammation in atherosclerosis remain largely unknown. Here, we identify the nuclear transcription factor c-Myb as an important mediator of atherosclerotic disease in mice. Atherosclerosis-prone animals fed a diet high in cholesterol exhibit increased levels of c-Myb in the bone marrow. Use of mice that either harbor a c-Myb hypomorphic allele or where c-Myb has been preferentially deleted in B cell lineages revealed that c-Myb potentiates atherosclerosis directly through its effects on B lymphocytes. Reduced c-Myb activity prevents the expansion of atherogenic B2 cells yet associates with increased numbers of IgM-producing antibody-secreting cells (IgM-ASCs) and elevated levels of atheroprotective oxidized low-density lipoprotein (OxLDL)-specific IgM antibodies. Transcriptional profiling revealed that c-Myb has a limited effect on B cell function but is integral in maintaining B cell progenitor populations in the bone marrow. Thus, targeted disruption of c-Myb beneficially modulates the complex biology of B cells in cardiovascular disease.
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http://dx.doi.org/10.1016/j.celrep.2019.04.090DOI Listing
May 2019

Publisher Correction: Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

Nat Immunol 2019 May;20(5):664

Toronto General Hospital Research Institute, University Health Network (UHN), Toronto, Canada.

In the version of this article initially published, the equal contribution of the third author was omitted. The footnote links for that author should be "Sara Nejat" and the correct statement is as follows: "These authors contributed equally: Sarah A. Dick, Jillian A. Macklin, Sara Nejat." The error has been corrected in the HTML and PDF versions of the article.
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http://dx.doi.org/10.1038/s41590-019-0363-8DOI Listing
May 2019

Self-renewing resident cardiac macrophages limit adverse remodeling following myocardial infarction.

Nat Immunol 2019 01 11;20(1):29-39. Epub 2018 Dec 11.

Toronto General Hospital Research Institute, University Health Network (UHN), Toronto, Canada.

Macrophages promote both injury and repair after myocardial infarction, but discriminating functions within mixed populations remains challenging. Here we used fate mapping, parabiosis and single-cell transcriptomics to demonstrate that at steady state, TIMD4LYVE1MHC-IICCR2 resident cardiac macrophages self-renew with negligible blood monocyte input. Monocytes partially replaced resident TIMD4LYVE1MHC-IICCR2 macrophages and fully replaced TIMD4LYVE1MHC-IICCR2 macrophages, revealing a hierarchy of monocyte contribution to functionally distinct macrophage subsets. Ischemic injury reduced TIMD4 and TIMD4 resident macrophage abundance, whereas CCR2 monocyte-derived macrophages adopted multiple cell fates within infarcted tissue, including those nearly indistinguishable from resident macrophages. Recruited macrophages did not express TIMD4, highlighting the ability of TIMD4 to track a subset of resident macrophages in the absence of fate mapping. Despite this similarity, inducible depletion of resident macrophages using a Cx3cr1-based system led to impaired cardiac function and promoted adverse remodeling primarily within the peri-infarct zone, revealing a nonredundant, cardioprotective role of resident cardiac macrophages.
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http://dx.doi.org/10.1038/s41590-018-0272-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6565365PMC
January 2019

Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control.

J Immunol 2017 09 7;199(6):2149-2157. Epub 2017 Aug 7.

Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario M5G 1L7, Canada.

Hypercholesterolemia is a key risk factor for atherosclerosis and leads to the uptake of native and oxidized low-density lipoprotein (oxLDL) by macrophages (Mϕs) and foam cell formation. Inflammatory processes accompany Mϕ foam cell formation in the artery wall, yet the relationship between Mϕ lipid loading and their response to inflammatory stimuli remains elusive. We investigated proinflammatory gene expression in thioglycollate-elicited peritoneal Mϕs, bone marrow-derived Mϕs and dendritic cells, and RAW264.7 cells. Loading with oxLDL did not induce peritoneal Mϕ apoptosis or modulate basal-level expression of proinflammatory genes. Upon stimulation of TLR4, the rapid induction of IFN-β was inhibited in cells loaded with oxLDL, whereas the induction of other proinflammatory genes by TLR4 (LPS), TLR3 (polyriboinosinic-polyribocytidylic acid), TLR2 (PamCSK), and TLR9 (CpG) remained comparable within the first 2 h. Subsequently, the expression of a subset of proinflammatory genes (e.g., IL-1β, IL-6, CCL5) was reduced in oxLDL-loaded cells at the level of transcription. This phenomenon was partially dependent on NF erythroid 2-related factor 2 (NRF2) but not on nuclear liver X receptors α and β (LXRα,β), peroxisome proliferator-activated receptor-γ (PPARγ), and activating transcription factor 3 (ATF3). LPS-induced NF-κB reporter activity and intracellular signaling by NF-κB and MAPK pathways were comparable in oxLDL-loaded Mϕs, yet the binding of p65/RelA (the prototypic NF-κB family member) was reduced at IL-6 and CCL5 promoters. This study revealed that oxLDL loading of Mϕs negatively regulates transcription at late stages of TLR-induced proinflammatory gene expression and implicates epigenetic mechanisms such as histone deacetylase activity.
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http://dx.doi.org/10.4049/jimmunol.1601363DOI Listing
September 2017

Paradoxical Suppression of Atherosclerosis in the Absence of microRNA-146a.

Circ Res 2017 Aug 21;121(4):354-367. Epub 2017 Jun 21.

From the Toronto General Hospital Research Institute, University Health Network, Ontario, Canada (H.S.C, R.B., A.L., Z.C., E.A.S., N.K., S.A.M., M.H., M.I.C., C.S.R., J.E.F.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada (H.S.C, R.B., A.L., Z.C., E.A.S., N.K., S.A.M., M.H., M.I.C., C.S.R., J.E.F.); Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, Toronto, Ontario, Canada (H.S.C, R.B., A.L., Z.C., E.A.S., N.K., M.H., M.I.C., C.S.R., J.E.F.); Institute for Cardiovascular Prevention, Ludwig-Maximilians-University Munich, Germany (M.N.-J., A.S.); INSERM, Unit 970, Paris Cardiovascular Research Center-PARCC, France (A.H., C.M.B.); University of Ottawa Heart Institute, Ontario, Canada (M.-A.N., M.G., K.J.R.); and Pharmacology and Nutritional Sciences, University of Kentucky, Lexington (L.C., T.L., R.E.T.).

Rationale: Inflammation is a key contributor to atherosclerosis. MicroRNA-146a (miR-146a) has been identified as a critical brake on proinflammatory nuclear factor κ light chain enhancer of activated B cells signaling in several cell types, including endothelial cells and bone marrow (BM)-derived cells. Importantly, miR-146a expression is elevated in human atherosclerotic plaques, and polymorphisms in the precursor have been associated with risk of coronary artery disease.

Objective: To define the role of endogenous miR-146a during atherogenesis.

Methods And Results: Paradoxically, (low-density lipoprotein receptor null) mice deficient in develop less atherosclerosis, despite having highly elevated levels of circulating proinflammatory cytokines. In contrast, cytokine levels are normalized in mice receiving wild-type BM transplantation, and these mice have enhanced endothelial cell activation and elevated atherosclerotic plaque burden compared with mice receiving wild-type BM, demonstrating the atheroprotective role of miR-146a in the endothelium. We find that deficiency of in BM-derived cells precipitates defects in hematopoietic stem cell function, contributing to extramedullary hematopoiesis, splenomegaly, BM failure, and decreased levels of circulating proatherogenic cells in mice fed an atherogenic diet. These hematopoietic phenotypes seem to be driven by unrestrained inflammatory signaling that leads to the expansion and eventual exhaustion of hematopoietic cells, and this occurs in the face of lower levels of circulating low-density lipoprotein cholesterol in mice lacking in BM-derived cells. Furthermore, we identify sortilin-1(), a known regulator of circulating low-density lipoprotein levels in humans, as a novel target of miR-146a.

Conclusions: Our study reveals that miR-146a regulates cholesterol metabolism and tempers chronic inflammatory responses to atherogenic diet by restraining proinflammatory signaling in endothelial cells and BM-derived cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.310529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542783PMC
August 2017

CCL19-CCR7-dependent reverse transendothelial migration of myeloid cells clears Chlamydia muridarum from the arterial intima.

Nat Immunol 2016 Nov 26;17(11):1263-1272. Epub 2016 Sep 26.

Toronto General Research Institute, University Health Network, Toronto, Canada.

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions.
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http://dx.doi.org/10.1038/ni.3564DOI Listing
November 2016

Macrophages and Dendritic Cells: Partners in Atherogenesis.

Circ Res 2016 Feb;118(4):637-52

From the Division of Advanced Diagnostics, Toronto General Research Institute, Peter Munk Cardiac Centre, University Health Network, Toronto, Ontario, Canada (M.I.C., C.S.R.); Departments of Laboratory Medicine and Pathobiology (M.I.C., C.S.R.) and Immunology (C.S.R.), University of Toronto, Toronto, Ontario, Canada; and Laboratory of Cellular Physiology and Immunology, Institut de Researches Cliniques de Montréal, Montréal, Québec, Canada (C.C.).

Atherosclerosis is a complex chronic disease. The accumulation of myeloid cells in the arterial intima, including macrophages and dendritic cells (DCs), is a feature of early stages of disease. For decades, it has been known that monocyte recruitment to the intima contributes to the burden of lesion macrophages. Yet, this paradigm may require reevaluation in light of recent advances in understanding of tissue macrophage ontogeny, their capacity for self-renewal, as well as observations that macrophages proliferate throughout atherogenesis and that self-renewal is critical for maintenance of macrophages in advanced lesions. The rate of atherosclerotic lesion formation is profoundly influenced by innate and adaptive immunity, which can be regulated locally within atherosclerotic lesions, as well as in secondary lymphoid organs, the bone marrow and the blood. DCs are important modulators of immunity. Advances in the past decade have cemented our understanding of DC subsets, functions, hematopoietic origin, gene expression patterns, transcription factors critical for differentiation, and provided new tools for study of DC biology. The functions of macrophages and DCs overlap to some extent, thus it is important to reassess the contributions of each of these myeloid cells taking into account strict criteria of cell identification, ontogeny, and determine whether their key roles are within atherosclerotic lesions or secondary lymphoid organs. This review will highlight key aspect of macrophage and DC biology, summarize how these cells participate in different stages of atherogenesis and comment on complexities, controversies, and gaps in knowledge in the field.
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http://dx.doi.org/10.1161/CIRCRESAHA.115.306542DOI Listing
February 2016

Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth.

Nat Immunol 2016 Feb 7;17(2):159-68. Epub 2015 Dec 7.

Department of Immunology, University of Toronto, Toronto, Ontario, Canada.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.
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http://dx.doi.org/10.1038/ni.3343DOI 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

The Neurorepellent Slit2 Inhibits Postadhesion Stabilization of Monocytes Tethered to Vascular Endothelial Cells.

J Immunol 2015 Oct 21;195(7):3334-44. Epub 2015 Aug 21.

Program in Cell Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario M5G 1X8, Canada; Institute of Medical Science, University of Toronto, Toronto, Ontario M5S 1A8, Canada;

The secreted neurorepellent Slit2, acting through its transmembrane receptor, Roundabout (Robo)-1, inhibits chemotaxis of varied cell types, including leukocytes, endothelial cells, and vascular smooth muscle cells, toward diverse attractants. The role of Slit2 in regulating the steps involved in recruitment of monocytes in vascular inflammation is not well understood. In this study, we showed that Slit2 inhibited adhesion of monocytic cells to activated human endothelial cells, as well as to immobilized ICAM-1 and VCAM-1. Microfluidic live cell imaging showed that Slit2 inhibited the ability of monocytes tethered to endothelial cells to stabilize their actin-associated anchors and to resist detachment in response to increasing shear forces. Transfection of constitutively active plasmids revealed that Slit2 inhibited postadhesion stabilization of monocytes on endothelial cells by preventing activation of Rac1. We further found that Slit2 inhibited chemotaxis of monocytes toward CXCL12 and CCL2. To determine whether Slit2 and Robo-1 modulate pathologic monocyte recruitment associated with vascular inflammation and cardiovascular disease, we tested PBMC from patients with coronary artery disease. PBMC from these patients had reduced surface levels of Robo-1 compared with healthy age- and sex-matched subjects, and Slit2 failed to inhibit chemotaxis of PBMC of affected patients, but not healthy control subjects, toward CCL2. Furthermore, administration of Slit2 to atherosclerosis-prone LDL receptor-deficient mice inhibited monocyte recruitment to nascent atherosclerotic lesions. These results demonstrate that Slit2 inhibits chemotaxis of monocytes, as well as their ability to stabilize adhesions and resist detachment forces. Slit2 may represent a powerful new tool to inhibit pathologic monocyte recruitment in vascular inflammation and atherosclerosis.
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http://dx.doi.org/10.4049/jimmunol.1500640DOI Listing
October 2015

ApoE attenuates atherosclerosis via miR-146a.

Circ Res 2015 Jun;117(1):3-6

From the Toronto General Research Institute, University Health Network, Toronto, Canada (J.E.F., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada (J.E.F., M.I.C.); and Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, Toronto, Canada (J.E.F., M.I.C.).

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http://dx.doi.org/10.1161/CIRCRESAHA.115.306733DOI Listing
June 2015

Differential role of an NF-κB transcriptional response element in endothelial versus intimal cell VCAM-1 expression.

Circ Res 2015 Jul 1;117(2):166-77. Epub 2015 Jun 1.

From the Vascular Research Division, Department of Pathology, Center for Excellence in Vascular Biology (D.S.M., P.O.D., V.M.D., T.C.) and Cardiovascular Division (J.P., J.D.B.), Brigham and Women's Hospital and Harvard Medical School, Boston, MA; Advanced Diagnostics Division, Toronto General Research Institute, University Health Network Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada (M.I., M.C., A.S., A.C.L., S.-N.Z., M.F.B., J.J.-B., M.I.C.); Department of Geriatric Medicine, Kyoto University Hospital, Kyoto, Japan (M.I.); Case Cardiovascular Research Institute, Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, OH (S.M.H.); and Department of Pathology, Children's Hospital and Harvard Medical School, Boston, MA (T.C.).

Rationale: Human and murine Vcam1 promoters contain 2 adjacent nuclear factor-κB (NF-κB)-binding elements. Both are essential for cytokine-induced transcription of transiently transfected promoter-reporter constructs. However, the relevance of these insights to regulation of the endogenous Vcam1 gene and to pathophysiological processes in vivo remained unknown.

Objective: Determine the role of the 5' NF-κB-binding element in expression of the endogenous Vcam1 gene.

Methods And Results: Homologous recombination in embryonic stem cells was used to inactivate the 5' NF-κB element in the Vcam1 promoter and alter 3 nucleotides in the 5' untranslated region to allow direct comparison of wild-type versus mutant allele RNA expression and chromatin configuration in heterozygous mice. Systemic treatment with inflammatory cytokines or endotoxin (lipopolysaccharide) induced lower expression of the mutant allele relative to wild-type by endothelial cells in the aorta, heart, and lungs. The mutant allele also showed lower endothelial expression in 2-week atherosclerotic lesions in Vcam1 heterozygous/low-density lipoprotein receptor-deficient mice fed a cholesterol-rich diet. In vivo chromatin immunoprecipitation assays of heart showed diminished lipopolysaccharide-induced association of RNA polymerase 2 and NF-κB p65 with the mutant promoter. In contrast, expression of mutant and wild-type alleles was comparable in intimal cells of wire-injured carotid artery and 4- to 12-week atherosclerotic lesions.

Conclusions: This study highlights differences between in vivo and in vitro promoter analyses, and reveals a differential role for a NF-κB transcriptional response element in endothelial vascular cell adhesion molecule-1 expression induced by inflammatory cytokines or a cholesterol-rich diet versus intimal cell expression in atherosclerotic lesions and injured arteries.
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http://dx.doi.org/10.1161/CIRCRESAHA.117.306666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758452PMC
July 2015

Endothelial cells suppress monocyte activation through secretion of extracellular vesicles containing antiinflammatory microRNAs.

Blood 2015 May 2;125(20):3202-12. Epub 2015 Apr 2.

Toronto General Research Institute, University Health Network, Toronto, ON, Canada; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada; Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, Toronto, ON, Canada;

The blood contains high concentrations of circulating extracellular vesicles (EVs), and their levels and contents are altered in several disease states, including cardiovascular disease. However, the function of circulating EVs, especially the microRNAs (miRNAs) that they contain, are poorly understood. We sought to determine the effect of secreted vesicles produced by quiescent endothelial cells (ECs) on monocyte inflammatory responses and to assess whether transfer of microRNAs occurs between these cells. We observed that monocytic cells cocultured (but not in contact) with ECs were refractory to inflammatory activation. Further characterization revealed that endothelium-derived EVs (EC-EVs) suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses. EVs isolated from mouse plasma also suppressed monocyte activation. Importantly, injection of EC-EVs in vivo repressed monocyte/macrophage activation, confirming our in vitro findings. We found that several antiinflammatory microRNAs were elevated in EC-EV-treated monocytes. In particular, miR-10a was transferred to monocytic cells from EC-EVs and could repress inflammatory signaling through the targeting of several components of the NF-κB pathway, including IRAK4. Our findings reveal that ECs secrete EVs that can modulate monocyte activation and suggest that altered EV secretion and/or microRNA content may affect vascular inflammation in the setting of cardiovascular disease.
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http://dx.doi.org/10.1182/blood-2014-11-611046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440888PMC
May 2015

Effect of disturbed blood flow on endothelial cell gene expression: a role for changes in RNA processing.

Arterioscler Thromb Vasc Biol 2014 Sep;34(9):1806-8

From the Toronto General Research Institute, University Health Network, and the Department of Laboratory Medicine and Pathobiology, University of Toronto (M.I.C.), and St Michael's Hospital, Division of Nephrology, Department of Medicine, University of Toronto (P.A.M.), Toronto, Canada.

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http://dx.doi.org/10.1161/ATVBAHA.114.304099DOI Listing
September 2014

MicroRNA-146 represses endothelial activation by inhibiting pro-inflammatory pathways.

EMBO Mol Med 2013 Jul 3;5(7):1017-34. Epub 2013 Jun 3.

Toronto General Research Institute, University Health Network, Toronto, Canada.

Activation of inflammatory pathways in the endothelium contributes to vascular diseases, including sepsis and atherosclerosis. We demonstrate that miR-146a and miR-146b are induced in endothelial cells upon exposure to pro-inflammatory cytokines. Despite the rapid transcriptional induction of the miR-146a/b loci, which is in part mediated by EGR-3, miR-146a/b induction is delayed and sustained compared to the expression of leukocyte adhesion molecules, and in fact coincides with the down-regulation of inflammatory gene expression. We demonstrate that miR-146 negatively regulates inflammation. Over-expression of miR-146a blunts endothelial activation, while knock-down of miR-146a/b in vitro or deletion of miR-146a in mice has the opposite effect. MiR-146 represses the pro-inflammatory NF-κB pathway as well as the MAP kinase pathway and downstream EGR transcription factors. Finally, we demonstrate that HuR, an RNA binding protein that promotes endothelial activation by suppressing expression of endothelial nitric oxide synthase (eNOS), is a novel miR-146 target. Thus, we uncover an important negative feedback regulatory loop that controls pro-inflammatory signalling in endothelial cells that may impact vascular inflammatory diseases.
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http://dx.doi.org/10.1002/emmm.201202318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3721471PMC
July 2013

α1β1 integrin-mediated adhesion inhibits macrophage exit from a peripheral inflammatory lesion.

J Immunol 2013 Apr 18;190(8):4305-14. Epub 2013 Mar 18.

Toronto General Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada.

Integrins are adhesion molecules critical for the recruitment of leukocytes from blood into peripheral tissues. However, whether integrins are also involved in leukocyte exit from peripheral tissues via afferent lymphatics to the draining lymph node remains poorly understood. In this article, we show that adhesion by the collagen IV-binding integrin α1β1 unexpectedly inhibited macrophage exit from inflamed skin. We monitored macrophages exiting mouse footpads using a newly developed in situ pulse labeling technique. Blockade of α1β1 integrin or genetic deletion (Itga1(-/-)) increased macrophage exit efficiency. Chemotaxis assays through collagen IV showed more efficient migration of Itga1(-/-) macrophages relative to wild type. Given that macrophages are key orchestrators of inflammation, α1β1 integrin adhesion may represent a mechanism for regulating inflammatory responses by controlling macrophage exit or persistence in inflamed tissues.
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http://dx.doi.org/10.4049/jimmunol.1202097DOI Listing
April 2013

Characterization of fibrinogen-like protein 2 (FGL2): monomeric FGL2 has enhanced immunosuppressive activity in comparison to oligomeric FGL2.

Int J Biochem Cell Biol 2013 Feb 2;45(2):408-18. Epub 2012 Nov 2.

Multi-Organ Transplant Program, Toronto General Hospital, University Health Network, University of Toronto, Toronto, Ontario, Canada M5G 2N2.

Fibrinogen-like protein 2 (FGL2), a novel effector molecule of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg), mediates its suppressive activity through binding to low affinity Fcγ receptors expressed on antigen presenting cells (APCs). FGL2 has been implicated in the pathogenesis of viral hepatitis, xeno- and allotransplant rejection, and rheumatoid arthritis. Here we fully analyzed the structure-function relationships of recombinant murine FGL2 generated in COS-7 cells and identified the receptor binding domains. Native FGL2 exists as an oligomer with a molecular weight of approximately 260 kDa, while under reducing conditions, FGL2 has a molecular weight of 65 kDa suggesting that native FGL2 is composed of four monomers. By site-directed mutation, cysteines at positions 94, 97, 184 and 187, found in the coiled-coil domain were shown to be crucial for FGL2 oligomerization. Monomeric FGL2 had a lower affinity binding to APCs, but increased immunosuppressive activity compared to oligomeric FGL2. Deglycosylation demonstrated that sugar moieties are critical for maintaining solubility of FGL2. SWISS-MODEL analysis suggested that FGL2 has a similar tertiary structure with other members of the fibrinogen family such as fibrinogen and tachylectin. Mutational analysis of cysteine residues and Western blots suggested an asymmetric bouquet-shaped quaternary structure for oligomeric FGL2, resembling many pattern-recognition molecules in the lectin pathway of innate immunity. The functional motifs of FGL2 were mapped to the C terminal globular domain, using a peptide blockade assay. These results collectively define the biochemical and immunological determinants of FGL2, an important immunosuppressive molecule of Treg providing important insights for designing FGL2-related therapeutics.
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http://dx.doi.org/10.1016/j.biocel.2012.10.014DOI Listing
February 2013

Taming endothelial activation with a microRNA.

J Clin Invest 2012 Jun 24;122(6):1967-70. Epub 2012 May 24.

Division of Cellular and Molecular Biology, Toronto General Research Institute, University Health Network, Toronto, Ontario, Canada.

Inflammation plays an essential role in vascular pathologies, including those associated with sepsis and atherosclerosis. Identifying negative regulators of inflammatory signaling pathways may provide novel therapeutic targets for these diseases. In this issue of the JCI, Sun et al. show that in endothelial cells, microRNA-18 1b (miR-18 1b) plays a vital role in controlling inflammation by targeting importin-α3, a regulator of NF-κB nuclear import. These findings provide compelling evidence that modulation of microRNAs may be a useful therapeutic approach for inflammatory vascular diseases.
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http://dx.doi.org/10.1172/JCI63818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3366417PMC
June 2012

Actin polymerization stabilizes α4β1 integrin anchors that mediate monocyte adhesion.

J Cell Biol 2012 Apr;197(1):115-29

Toronto General Research Institute, University Health Network, Toronto, M5G 2C4 Ontario, Canada.

Leukocytes arrested on inflamed endothelium via integrins are subjected to force imparted by flowing blood. How leukocytes respond to this force and resist detachment is poorly understood. Live-cell imaging with Lifeact-transfected U937 cells revealed that force triggers actin polymerization at upstream α4β1 integrin adhesion sites and the adjacent cortical cytoskeleton. Scanning electron microscopy revealed that this culminates in the formation of structures that anchor monocyte adhesion. Inhibition of actin polymerization resulted in cell deformation, displacement, and detachment. Transfection of dominant-negative constructs and inhibition of function or expression revealed key signaling steps required for upstream actin polymerization and adhesion stabilization. These included activation of Rap1, phosphoinositide 3-kinase γ isoform, and Rac but not Cdc42. Thus, rapid signaling and structural adaptations enable leukocytes to stabilize adhesion and resist detachment forces.
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http://dx.doi.org/10.1083/jcb.201107140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3317807PMC
April 2012

Talin-1 and kindlin-3 regulate alpha4beta1 integrin-mediated adhesion stabilization, but not G protein-coupled receptor-induced affinity upregulation.

J Immunol 2011 Oct 12;187(8):4360-8. Epub 2011 Sep 12.

Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L7, Canada.

Chemokine/chemoattractant G protein-coupled receptors trigger an inside-out signaling network that rapidly activates integrins, a key step in inflammatory leukocyte recruitment. Integrins mediate leukocyte arrest and adhesion to endothelium through multivalent binding, and they transmit outside-in signals to stabilize adhesion and coordinate cell spreading and migration. In the present study, we used RNA interference in the U937 monocytic cell line to investigate the role of talin-1, kindlin-3, and α-actinin-1 in the fMLF- and SDF-1α-induced upregulation of α(4)β(1) integrin affinity and consequent adhesive events. Affinity upregulation of α(4)β(1) integrin was not impaired by small interfering RNA knockdown of talin-1, kindlin-3, or α-actinin-1. Only kindlin-3 knockdown increased flow-induced detachment from VCAM-1-coated surfaces in response to fluid flow, whereas knockdown of either talin-1 or kindlin-3 increased detachment from ICAM-1-coated surfaces. Biochemical analyses revealed that α(4)β(1) expression was highly enriched in U937 cell microridges and murine lymphocyte microvilli. Kindlin-3 was present throughout the cell, whereas talin-1 was largely excluded from microridges/microvilli. The subcellular colocalization of α(4)β(1) and kindlin-3 in microridges may explain why kindlin-3 rapidly associates with α(4)β(1) after G protein-coupled receptor signaling and contributes to adhesion strengthening. Talin-1 contributed to α(4)β(1)-dependent chemotaxis, suggesting that it participates in a later stage of the leukocyte adhesion cascade when the leukocyte cytoskeleton undergoes dramatic rearrangement.
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http://dx.doi.org/10.4049/jimmunol.1003725DOI Listing
October 2011

Resident intimal dendritic cells and the initiation of atherosclerosis.

Curr Opin Lipidol 2010 Oct;21(5):397-403

Toronto General Research Institute, University Health Network, University of Toronto, Toronto, Ontario, Canada.

Purpose Of Review: To highlight the fact that regional differences in the normal arterial intima are critical to atherosclerotic lesion formation driven by systemic risk factors.

Recent Findings: At arterial curvatures, bifurcations and branches unique hemodynamics influence endothelial cell signaling and gene expression patterns, which create a proinflammatory environment, with low-grade recruitment of monocytes and accumulation of cells with dendritic features in the intima. Upon induction of hypercholesterolemia, these resident intimal dendritic cells initiate atherosclerosis by rapidly engulfing lipid and becoming the first foam cells in nascent lesions. This step precedes endothelial cell activation and increased monocyte recruitment.

Summary: The unique features of the arterial intima at atherosclerosis-susceptible sites do not lead to disease under normal physiological conditions, but this intimal environment promotes the initiation of atherogenesis upon induction of systemic risk factors such as hypercholesterolemia.
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http://dx.doi.org/10.1097/MOL.0b013e32833ded96DOI Listing
October 2010

Increased oxidative stress in atherosclerosis-predisposed regions of the mouse aorta.

Life Sci 2010 Jul 8;87(3-4):100-10. Epub 2010 Jun 8.

University of Texas Health Science Center at Houston and Texas Heart Institute, Houston, TX, USA.

Aims: The localization of atherosclerotic lesions to predictable regions in mammalian arteries has been recognized for over a century. We sought to investigate the association between oxidative stress and regional susceptibility of the mouse aorta to atherosclerosis.

Main Methods: En face confocal microscopy was employed to assess oxidative stress in the aortic intima of atherosclerosis-susceptible and protected regions of wild-type C57BL/6 mouse. Expression of reactive oxygen species and antioxidant producing genes were compared in endothelial cells from the susceptible and protected regions.

Key Findings: In vivo administration of redox-sensitive fluorescent dyes revealed an increase in the production of reactive oxygen species (ROS) in the atherosclerosis-susceptible regions relative to the protected regions. In contrast, Hoechst a redox-insensitive dye distributed evenly in the susceptible and protected regions. Accumulation of superoxide in the susceptible regions of the aorta was significantly blocked by the administration of Diphenyleneiodonium, a flavoprotein inhibitor. mRNA levels of superoxide-producing and scavenging enzymes were significantly increased in the regions predisposed to atherosclerosis. The regional difference in oxidative stress was at a lesser magnitude in BALB/c than the atherosclerosis-susceptible mouse (C57BL/6).

Significance: Our study for the first time demonstrated an augmented oxidative stress in atherosclerosis-susceptible regions of the normal mouse aorta.
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http://dx.doi.org/10.1016/j.lfs.2010.05.016DOI Listing
July 2010

Aortic regurgitation dramatically alters the distribution of atherosclerotic lesions and enhances atherogenesis in mice.

Arterioscler Thromb Vasc Biol 2010 Jun 18;30(6):1181-8. Epub 2010 Mar 18.

Mouse Imaging Centre, The Hospital for Sick Children, Toronto, Ontario, Canada.

Objective: Hemodynamics plays a critical role in atherogenesis, but the association between flow pattern and preferential localization of lesion is not fully understood. We developed a mouse model of aortic valve regurgitation (AR) to change the aortic flow pattern and observed the effects on plaque formation.

Methods And Results: High-frequency Doppler ultrasound imaging of 10 untreated C57BL/6J mice and 6 sham-treated low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice revealed consistent antegrade blood flow throughout the aorta and oscillatory flow only along the lesser curvature of the aortic arch. Catheter-induced AR in 7 Ldlr(-/-) mice produced various degrees of diastolic retrograde flow throughout the aorta. After the mice were fed a cholesterol-enriched diet for 6 weeks, the burden of atherosclerotic lesions was increased 6-fold, with the naturally plaque-resistant descending aorta becoming susceptible. The AR severity correlated positively with the lesion burden in the descending thoracic and abdominal aorta but negatively with the lesions in the ascending aorta and aortic arch.

Conclusions: This AR model is valuable for elucidating the relationship between hemodynamics and predisposition of the artery wall to atherosclerosis, because of the significant alterations in local flow patterns and the conversion of large regions in the descending aorta from lesion resistant to lesion prone.
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http://dx.doi.org/10.1161/ATVBAHA.110.204198DOI Listing
June 2010