bluuebunny/crossref_metadata_2025 huggingface dataset:
Da arte de governar bem ou mal: A necessidade do Estado e o exercício da justiça no alvorecer das Minas
Authors: Pereira Marcos Aurélio
Published: March 2019

O artigo aborda aspectos da cultura política no Império ultramarino português e o exercício do poder dos governadores enquanto alter ego do monarca na América portuguesa. As análises privilegiam a política da Coroa no século XVIII, percebendo uma transformação das formas de governar através das distintas práticas de estabelecer justiça. Incide também sobre o exame das mercês régias como fontes de justiça distributiva conferidas às elites reinóis. Examinam-se as estratégias político-administrativas do conde de Assumar, governador das Minas Gerais entre 1717 e 1721, cujo governo oscilou, segundo as fontes, entre o bom e o mau governante. Uma nova percepção da razão de Estado em transformação emerge na região mineradora que se expressa no controle da população, no enfrentamento dos poderosos locais, apaziguando contendas e estabelecendo a ordem. A implementação dessa política alterou, paulatinamente, os pressupostos da arte de governar em função de uma incipiente racionalidade administrativa por parte da Coroa.
Identidade étnica ou Identidades étnicas? A italianidade em Oswaldo Truzzi
Authors: Almeida Renata Geraissati Castro de
Published: March 2019

Resenha do livro: TRUZZI, Oswaldo Mario Serra. Italianidade no interior paulista – percursos e descaminhos de uma identidade étnica (1880-1950).  São Paulo: Editora Unesp, 2016.

O livro Italianidade no interior paulista: percursos e descaminhos de uma identidade étnica (1880-1950) publicado em 2016, pelo professor da Universidade Federal de São Carlos, Oswaldo Truzzi, tem por objetivo compreender como se deu o processo de estruturação de uma identidade étnica do grupo de indivíduos que emigraram da Itália, no recorte temporal que abrange dos anos de 1880 a 1950. O autor, com uma relevante carreira nos estudos migratórios, elenca três ocasiões que se constituíram em marcos para uma mudança neste sentimento. São elas: os momentos iniciais da imigração e a construção da “italianidade” fora da Itália; a emergência do fascismo na Itália; e o Estado Novo. Ao perscrutar as diversas camadas sobrepostas na identidade italiana, visou mostrá-la como um processo histórico constantemente negociado, em que ora ocorre a aceitação, ora a resistência à assimilação. A pertinência de seu livro está em historicizar o termo “italianidade” mostrando seus desafios e particularidades em diferentes temporalidades.
A teologia natural na cultura científica da Ilustração portuguesa: Oratorianos e Franciscanos (1750-1800)
Authors: Ferreira Breno Ferraz Leal
Published: March 2019

O objetivo deste artigo é refletir como a história natural foi mobilizada para combater as doutrinas materialistas ilustradas em Portugal na segunda metade do século XVIII. Propõe-se analisar o pensamento de três importantes homens de ciência portugueses que se valeram da teologia natural para defender, por diferentes meios, a validade da doutrina católica: o padre oratoriano Teodoro de Almeida e os frades franciscanos Manuel do Cenáculo e José Mayne. Argumentaremos que, naquele contexto da Ilustração, tais intelectuais enxergaram a necessidade de se opor publicamente às “novas filosofias do século”, sempre buscando fundamentar a legitimidade da doutrina católica por sua racionalidade.
A emigração como força civilizadora: portugueses nas colônias africanas e no Brasil independente
Authors: Gonçalves Paulo Cesar
Published: March 2019

A análise de boletins da Sociedade de Geografia de Lisboa, dos inquéritos parlamentares sobre a emigração e de estudos de alguns pensadores contemporâneos permite focalizar os movimentos migratórios ultramarinos, inserindo-os na expansão colonialista portuguesa com propósitos específicos de configuração do império em África entre o final do século XIX e o início do XX. O objetivo é investigar a defesa da emigração de portugueses para as possessões africanas como alternativa ao processo de colonização em contraponto ao tradicional destino brasileiro, com suas perspectivas econômicas individuais e nacionais representadas pelas remessas.
Marcello Caetano: o ethos intelectual e as artimanhas do poder
Authors: Secco Lincoln
Published: March 2019

Resenha do livro: MARTINHO, Francisco Carlos Palomanes. Marcello Caetano: Uma biografia (1906-1980). Lisboa: Objectiva, 2016.
Os africanos escolhem o que vão levar: os tecidos indianos no comércio de marfim e escravos
Authors: Folador Thiago de Araujo
Published: March 2019

Resenha do livro: MACHADO, Pedro. Ocean of trade: South Asian merchants, Africa and the Indian Ocean, c. 1750–1850 . Cambridge: Cambridge University Press, 2014. 315 p.
A Escravidão na imprensa: política antiescravista e escravista no Rio de Janeiro, século XIX
Authors: Oliveira Felipe Garcia de
Published: March 2019

O texto é uma resenha da obra: YOUSSEF, Alain El. Imprensa e Escravidão. Política e tráfico negreiro no Império do Brasil (Rio de Janeiro, 1822- 1850) São Paulo: Intermeios/Fapesp, 2016.
Activity patterns of the small and medium felid (Mammalia: Carnivora: Felidae) guild in northeastern India
Authors: Mukherjee Shomita , Singh Priya , Silva André Pinto , Ri Chandan , Kakati Kashmira , Borah Binod , Tapi Tana , Kadur Sandesh , Choudhary Prafull , Srikant Shikha , Nadig Surabhi , Navya R. , Björklund Mats , Ramakrishnan Uma
Published: March 2019

Fifteen extant species of cats inhabit India, and the northeastern region of the country is among the richest with nine species.  Among these are the “standard four”, an assemblage of Clouded Leopard Neofelis nebulosa, Asiatic Golden Cat Catopuma temminckii, Marbled Cat Pardofelis marmorata, and Leopard Cat Prionailurus bengalensis, which also occur across southeastern Asia.  Within India, despite several surveys in this region, very little information exists on the ecology of this assemblage to explain their co-occurrence.  In this paper, we put together data from several independent camera trapping studies over 10 sites across northeastern India to examine and interpret diel activity patterns of this group.  While we present results for all the four species, we focus on two species, the Marbled Cat and Leopard Cat, which are of very similar body size and are potential competitors.  We used kernel density estimates to measure diel activity patterns of all four species and overlap in activity between Marbled Cat and Leopard Cat at the regional scale as well as the point scale.  We obtained 783 captures of the standard four from >27,500 trap nights.  The Asiatic Golden Cat and Marbled Cat were strongly diurnal, Clouded Leopard largely crepuscular and nocturnal, and Leopard Cat largely nocturnal.  The degree of overlap between Marbled Cat and Leopard Cat activity was low and in consensus with other studies across southeastern Asia.  We interpret this as the differing niche spaces of the two cats due to their specific pre-existing adaptations, not restricted to the effects of competition.  The point scale analysis when both cats are captured at the same location and separately show no shift in activity pattern, supporting our hypothesis of pre-existing differences in resources, such as food, playing a major role in facilitating co-existence.  Our study, however, is preliminary and additional information with robust analysis is required to test this finding.
Abstract 003: The Impact of MHCII Loss in Myeloid Cells and Adipocytes on Atherosclerosis and Liver Fat Accumulation
Authors: Blaszczak Alecia , Wright Valerie , Liu Joey , Deng Tuo , Bradley David , Bergin Stephen , Hsueh Willa
Published: March 2019

The adipocyte is capable of functioning as an antigen presenting cell in the setting of diet induced obesity in both mouse models and humans; this adipocyte activity instigates adipose tissue (AT) inflammation. To determine the relative roles of adipocyte vs. myeloid adaptive immunity on obesity-associated complications, we investigated myeloid vs. adipocyte-specific knockout of a major histocompatibility II (MHCII) protein, H2Ab1, which in mice prevents antigen presentation. Loss of adipocyte, but not myeloid, MHCII led to improvements in insulin sensitivity, adipose tissue inflammation and inhibited the shift from anti to pro-inflammatory immune cells in adipose tissue during high fat diet feeding. Both models were placed on a pro-atherogenic background (LDLR -/-), then aged for one year to accelerate atherosclerosis and were fed a western diet high in cholesterol and fat for 12 weeks. Adipocyte specific, but not myeloid specific, loss of MHCII in LDLR -/- led to dramatic improvements in atherosclerosis development, liver fat accumulation and insulin sensitivity in conjunction with an increase in the adipose tissue Tregs and a decrease in the pro-inflammatory adipocyte cytokine gene expression with no differences noted in body weight or fat. Whereas in LDLR -/- with lack of myeloid MHCII compared to LDLR -/- , we saw no differences in liver fat accumulation, atherosclerosis development or insulin sensitivity with a decrease in the anti-inflammatory Tregs, a comparable increase in adipocyte pro-inflammatory cytokine expression, but a decrease in the pro-inflammatory M1 like macrophages. These studies highlight the importance of the adipocyte as a functional contributor to the development of obesity related complications and provide a potential targeted therapeutic avenue in the management of these metabolic diseases.
Abstract 004: Comprehensive Assessment of Immune Cells in Mouse and Human Atherosclerosis by Single-cell RNA-sequencing and Mass Cytometry
Authors: Winkels Holger , Ehinger Erik , Vassalo Melanie , Buscher Konrad , Dinh Huy , Kobiyama Kouji , Hamers Anouk , Cochain Clement , Vafadarnejad Ehsan , Saliba Antoine E , Zernecke Alma , Bala Pramod A , Ghosh Amlan K , Michel Nathaly A , Hoppe Natalie , Hilgendorf Ingo , Zirlik Andreas , Hedrick Catherine , Ley Klaus , Wolf Dennis
Published: March 2019

Atherosclerosis, an inflammatory disease of large arteries, is - through its clinical manifestations stroke and myocardial infarction - globally the leading cause of morbidity and mortality. The interplay of pro- and anti-inflammatory leukocytes in the aorta modulates and drives atherosclerosis. Although cells of the innate and adaptive immune system are found in atherosclerotic plaques, their phenotypic and functional diversity is poorly understood. Here, we applied single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to assess leukocyte diversity in depth, thus defining an immune cell atlas in atherosclerosis. Single cell transcriptional profiling of aortic leukocytes from 20-week old chow (CD) and western diet (WD) fed
Apoe
-/-
and
Ldlr
-/-
mice revealed 11 phenotypically different leukocyte clusters. Atherosclerotic aortas exhibited enhanced leukocyte diversity, whilst WD further changed the abundance of leukocyte subpopulations. Gene set enrichment analysis of single cells established that multiple pathways, e.g. for lipid metabolism, proliferation, and cytokine secretion, pertained to particular leukocyte clusters. Applying a novel 35-marker CyTOF panel with metal-labelled antibodies confirmed the phenotypic diversity of aortic leukocytes. Among lymphocytes, we detected three principal B-cell subsets defined by scRNAseq, CyTOF, and flow cytometry. These B cell subsets harbor distinct surface marker expression, functional gene pathways, and
ex vivo
cytokine production. Finally, we used leukocyte cluster gene signatures to assess leukocyte frequencies in 121 human plaques by a transcriptomic deconvolution strategy. This approach revealed a similar immune cell complexity in human carotid plaques with a higher percentage of monocytes and macrophages. In addition, the frequency of genetically defined leukocyte populations in carotid plaques predicted cardiovascular events in patients. The definition of leukocyte diversity by high-dimensional analyses enables a fine-grained analysis of aortic leukocyte subsets, reveals new immunological mechanisms and cell-type specific pathways, and may result in novel diagnostic risk stratification tools.
Abstract 008: Perivascular Cell-specific Knockout of the Stem Cell Pluripotency Gene Oct4 Inhibits Angiogenesis in Part by Attenuating Perivascular and Endothelial Cell Migration
Authors: Hess Daniel L , Kelly-Goss Molly R , Cherepanova Olga , Nguyen Anh T , Annex Brian H , Peirce Shayn M , Owens Gary K
Published: March 2019

Objective:
Angiogenesis requires coordinated migration of endothelial cells (EC) and perivascular cells, including smooth muscle cells and pericytes (SMC-P). Perivascular cell-specific mechanisms by which SMC-P migrate and invest EC remain largely unknown. Herein, we used Myh11-CreER
T2
SMC-P lineage tracing, combined with SMC-P specific knockout of the stem cell pluripotency gene Oct4, to test the hypothesis that SMC-P derived Oct4 regulates perivascular cell migration and recruitment necessary for angiogenesis.

Methods and Results:
Myh11-CreER
T2
ROSA floxed STOP eYFP Oct4
WT/WT
and Myh11-CreER
T2
ROSA floxed STOP eYFP Oct4
FL/FL
littermate mice were injected with tamoxifen from 6-8 weeks of age to permanently label Myh11-expressing cells with eYFP, without or with Oct4 KO, respectively. Mice were subjected to either corneal alkali burn or hindlimb ischemia (HLI), monitored by live confocal imaging and laser doppler perfusion respectively, and sacrificed for tissue analysis. SMC-P Oct4 KO resulted in markedly impaired eYFP+ (SMC-P derived) migration and increased vascular leak following corneal alkali burn. EC neovascular area and migration distance were also significantly decreased in SMC-P specific Oct4 KO mice. Following HLI, SMC-P Oct4 KO mice had impaired perfusion recovery and decreased capillary density. Slit3 was expressed in eYFP+ cells of the microvasculature in both cornea and muscle and was downregulated following Oct4 KO. RNA-seq and qRT-PCR analysis of cultured SMC revealed dysregulation of Slit3 as well as additional members of the Slit-Robo pathway of guidance genes, including downregulation of the receptors Robo1 and Robo2 and upregulation of Slit2 following loss of Oct4.

Conclusions:
Taken together, we demonstrate that SMC-P derived Oct4 is essential for angiogenesis in both corneal alkali burn and HLI models. These effects are at least partially due to Oct4-dependent regulation of the Slit-Robo pathway in SMC-P, with Oct4 KO resulting in dysregulated migration of both EC and SMC-P. To our knowledge, this is the first direct evidence that loss of a single gene exclusively in SMC-P impacts angiogenesis following injury.
Abstract 013: Genotype to Phenotype: Function of Rare Coding Variants in
ANGPTL3
Authors: Wang Xiao , Vourakis A. Christina , Sperry Alexandra E , Chadwick Alexandra C , Li Wenjun , Lv Wenjian , Musunuru Kiran
Published: March 2019

Angiopoietin-like 3 (
ANGPTL3
) associates strongly with blood lipid phenotypes in human genetics studies and has thus emerged as promising therapeutic target for plasma lipids. We examined the coding regions of
ANGPTL3
and identified 82 rare variants in individuals from separate human cohorts. In light of the seemingly favorable clinical consequences of
ANGPTL3
deficiency, we established an experimental framework to determine the functional consequences of rare
ANGPTL3
missense mutations
in vivo
. We generated an
Angptl3
knockout mouse, which exhibited decreased triglyceride (TG) (61%, P < 0.001) and decreased total cholesterol (31%, P < 0.002). We attempted to rescue this phenotype using adenoviruses expressing either wild-type or missense variant of
ANGPTL3
. To date, all 82 rare missense variants have been assessed, of which 25 were validated in terms of loss-of-function as severe (conferring <25% of wild-type activity as assessed by either TG or cholesterol levels), 18 were moderate (25-50%), 18 were mild (50-75%), and 20 were benign (75-125%) while 1 was gain-of-function (>125%), underscoring the need for functional characterization of variants of uncertain significance. Furthermore, we noticed that 4 variants (H343R, S86L, E129G and T256K) were loss-of-function on triglyceride levels, while benign on cholesterol levels. On the other hand, 7 variants (L75F, C408R, Y350H, K387Q, Q293E, D455E and M259R) were loss-of-function on cholesterol while benign on TG. ANGPTL3 has been implicated in regulating TG and cholesterol through different mechanisms. Therefore, we studied the potential mechanisms by knocking in variants into induced pluripotent stem cells (iPSCs)
in vitro
. We next differentiated edited cells into hepatocyte-like cells (HLCs). We observed no cleaved N-terminal fragment of ANGPTL3 in HLCs with the E129G variant while the expression level of full-length ANGPTL3 was unaffected. It has been reported that N-terminal fragment is sufficient to inhibit lipoprotein lipase activity which is the primary mechanism by which triglyceride-rich lipoproteins are cleared from the circulation. These results indicate that variant E129G could not be cleaved and this might be the mechanism by which it only affects TG, not cholesterol levels.
Abstract 015: PAR4 Ala120Thr Variant Alters PAR4 Desensitization, Sensitivity to Platelet Antagonists and Risk of Large Vessel Stroke
Authors: Whitley Michael , Henke David , Simon Lukas , Vesci Joanne , Holinstat Michael , Nieman Marvin , Shaw Chad , Bray Paul , Edelstein Leonard
Published: March 2019

F2RL3
encodes protease-activated receptor 4 (PAR4) and harbors a A/G SNP (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP), AYPGKF, which does not occur in vivo. Platelet function studies from healthy human donors demonstrated that compared to rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation to low concentrations (<1 nM) of thrombin, increased Ca
2+
signaling, increased granule secretion and decreased PAR4 desensitization. The increased thrombin sensitivity of rs773902 AA platelets required both PAR4 and PAR1. Whereas receptor cleavage, dimerization, or lipid raft localization were not different between platelets from the two
F2RL3
genotypes, rs773902 AA platelets required more than 3-fold amounts of PAR4-AP for receptor desensitization. PAR4-AP stimulation of PAR4-transfected COS-7 cells
increased
PAR4 levels on the cell surface. Thrombin-stimulated platelets from rs773902 AA donors displayed reduced inhibition by P2Y12 and PAR1 blockers, but not aspirin. Finally, the association of rs773902 alleles with stroke was assessed using the Stroke Genetics Network (SiGN) study. Patients carrying at least one copy of the rs773902 A allele had an increased risk of both large vessel stroke and diabetes in the SiGN dataset. In summary, both PAR4 and PAR1 are required for platelet activation to sub-nanomolar thrombin concentrations. Compared to rs773902 GG platelets, rs773902 AA platelets have a lower EC50 for thrombin and are relatively resistant to receptor desensitization, perhaps due to anterograde trafficking of internal PAR4 to the plasma membrane post-stimulation. The association of the rs773902 A variant with large vessel stroke, diabetes and partial resistance to PAR1 and P2Y12 inhibition must be considered for development of personalized anti-platelet agents.
Abstract 022: Hepatic Sortilin Regulates Apolipoprotein B Secretion only Under Conditions of Secretory StressDependent of the Presence of Secretory Stress
Authors: Conlon Donna M , Rodrigues Amrith , Guo Kathy , Hand Nicholas , Rader Daniel
Published: March 2019

SORT1
is strongly associated with plasma lipid traits and coronary artery disease. The protein it encodes, sortilin is a multi-ligand receptor involved in trafficking of proteins from the Golgi to the lysosome. Hepatic sortilin overexpression reduces VLDL secretion, but the effects of loss of sortilin function on apolipoprotein B100 (apoB) and VLDL secretion have been contradictory and perplexing. Conflicting studies have variously reported increased and decreased apoB/VLDL secretion in response to loss of sortilin, but used a variety of different models and methods of ablating sortilin function. To resolve this, we first measured VLDL secretion in Sort1 knockout (KO) mice on a chow diet and observed no differences in either apoB or TG secretion. ApoB secretion was similarly unaffected in Sort1 KO primary hepatocytes or in McA-RH7777 cells treated with siRNA to knockdown sortilin. We concluded that in a basal secretory state, loss of sortilin alone does not impact apoB secretion, leading us to test if differences in secretory state might explain the discrepancies in published results. We found that when hAPOB was overexpressed, Sort1 knockdown further increased its secretion. The reports of increased VLDL secretion with Sort1 knockout occurred with apoB overexpression, or on high fat diet (HFD); consistent with our hypothesis that secretory overload or metabolic stress are required to uncover the deficiency phenotype. We placed Sort1 KO mice on a 45% HFD for 12 weeks and observed a significant increase in VLDL secretion compared to controls. In addition, when we lipid-loaded hepatocytes with oleic acid or palmitic acid, apoB secretion was increased, and Sort1 knockdown further increased this compared to control. We treated cells with Tunicamycin to induce ER stress, and found that apoB secretion was decreased in the control group was observed, but not in the absence of Sort1, suggesting that sortilin regulates apoB secretion the cell is stressed. Based on these data, we propose that hepatic sortilin regulates the post-ER fate of apoB for either degradation or export; thereby coordinating intracellular apoB metabolism in response to the number and quality of apoB particles that reach the Golgi, and the level of post-ER pre-secretory proteolysis activity.
Abstract 031: A Ligand-specific Blockade of the Integrin Mac-1 Selectively Targets Pathologic Vascular Inflammation While Maintaining Protective Host-defense
Authors: Wolf Dennis , Anto Michel Nathaly , Buscher Konrad , Ley Klaus , Libby Peter , Bode Christoph , Peter Karlheinz , Zirlik Andreas
Published: March 2019

Background:
Integrins, such as Mac-1 (alphaMbeta2, CD11b/CD18), drive myeloid cell recruitment and inflammation in cardiovascular disease. Although integrins have generated interest as therapeutic targets in acute and chronic inflammation, they also contribute to host defense, regeneration, and haemostasis. To overcome these limitations, we have designed a novel antibody that targets a distinct region within the Mac-1 major ligand-binding (I-) domain required for binding of its ligand CD40L. Here, we describe the consequences of a ligand-specific inhibition and its efficacy in mice.

Methods and Results:
To generate an antibody specifically targeting the interaction of Mac-1 with CD40L, mice were immunized with a peptide containing the CD40L-binding motif EQLKKSKTL. Antibody clones were screened and tested for specificity. One clone, termed anti-M7, showed specific peptide binding and blocked the binding of the activated, open conformation of Mac-1 to CD40L, but not to alternative Mac-1 ligands suggesting ligand specificity. Anti-M7 highly effectively limited leukocyte recruitment in vitro and to the mesentery and the omentum in intravital confocal microscopy. While conventional anti-Mac-1 antibodies induced outside-in activation of the integrin, anti-M7 did not provoke MAP-kinase dependent activation and expression of inflammatory cytokines in macrophages. Following cecal-ligation and puncture (CLP) sepsis in mice, a conventional anti-Mac-1 antibody inhibited leukocyte accumulation, but potentiated bacteremia and limited overall survival. In contrast, anti-M7 limited peritoneal leukocyte accumulation, reduced bacterial titers, and improved survival during CLP.

Conclusion:
We present proof-of-concept demonstrating selective inhibition of the interaction between the leukocyte integrin Mac-1 and CD40L by a novel monoclonal antibody. This antibody shows several advantages over conventional anti-integrin therapy, and prevented inflammation and sepsis-related mortality in mice. This novel approach gives new insight into ligand-specific integrin pathways and merits further evaluation as a selective anti-inflammatory therapy in cardiovascular disease.
Abstract 034: Leveraging Mouse Liver Co-expression Networks and Human Lipid Gwas Data to Identify Cholesterol Metabolism Genes
Authors: Li Zhonggang , Nguyen Jenny , Belisle Sabrina , Ly Sophia , Leyva Jaimes Fernanda , Parks Brian W
Published: March 2019

Genetic factors play an important role in contributing to variation in plasma lipid levels across the human population. Large genome-wide association studies (GWAS) in humans have identified more than 100 loci significantly associated with plasma levels of low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, total cholesterol (TC), and triglycerides. To prioritize genes that are involved in cholesterol metabolism, we developed a systematic approach to leverage genome-wide liver transcriptomic and proteomic data from multiple mouse reference populations along with human lipid GWAS data. We constructed global co-expression networks from twelve distinct mouse liver datasets, encompassing more than 800 unique mice and identified a conserved module of genes highly enriched for cholesterol biosynthesis. Based on replication across datasets and presence in transcript and protein, we prioritized 112 unique genes. Intersection of these 112 prioritized genes with human GWAS data for LDL, HDL, TC, and triglycerides identified 54 genes to be within 100 kilobases of a significant or suggestive significant single nucleotide polymorphism (SNP). Out of the 54 identified genes that overlap with human GWAS data, 29 have well documented biological roles in cholesterol metabolism, such as
LDLR
,
PCSK9
, and
INSIG1
. With the 25 identified genes with no described role in cholesterol metabolism, we tested for transcriptional regulation to cholesterol and performed a functional screen by siRNA knockdown. From this analysis, we identified twelve genes that show transcriptional regulation to cholesterol levels and nine genes that are able to modulate cholesterol metabolism when targeted
in vitro
with siRNA. Five genes out of the 25 prioritized genes show both transcriptional regulation to cholesterol and ability to modulate cholesterol metabolism in vitro. One of these genes,
Sestrin1
, we validate
in vivo
and
in vitro
as a modulator of cholesterol metabolism. Collectively, through a systematic approach we have identified 25 highly prioritized genes that have no documented role in cholesterol metabolism, five genes are transcriptionally regulated by cholesterol and influence cholesterol metabolism.
Abstract 038: Inflammasome Activation Triggers Blood Coagulation Through Pyroptosis
Authors: Wu Congqing , Wei Yinan , Li Zhenyu
Published: March 2019

Background:
Disseminated intravascular coagulation (DIC) is a frequent and fatal complication of sepsis. The presence of bacterial virulence factors can induce blood coagulation, yet the molecular events linking bacteria sensing to initiation of the coagulation cascade remain largely unknown. Inflammasome is the large sensory protein complex in the cytosol that alerts host to bacterial infection. The role of inflammasome in sepsis remains unclear.

Methods and Results:
We used
E. coli
type III secretion system (T3SS) inner rod protein EprJ and LPS to investigate the role of canonical and noncanonical inflammasome activation in sepsis, respectively. For efficient cytosolic delivery, EprJ was fused to the cytosolic translocation domain of anthrax lethal factor (LFn). Binding with LFn, anthrax protein protective agent (PA) delivers the LFn-EprJ fusion protein into the cytoplasma through receptor-mediated endocytosis. We found that EprJ, the inner rod protein, activated caspase-1 and induced systemic coagulation, while lipopolysaccharide (LPS) produced similar effects via caspase-11 activation. We also identified gasdermin D (Gsdmd) driven macrophage pyroptosis as a mechanism for the release of tissue factor, an essential initiator of coagulation cascades. Genetic deficiency of Gsdmd abolishes inflammasome-mediated coagulation.

Conclusion:
Inflammasome activation is a trigger for coagulation induced by Gram-negative bacterial products, connecting inflammation and thrombosis.
Abstract 045: SR-BI and PCPE2 Modulate Lipid Trafficking in Adipocytes
Authors: Xu Hao , Kaul Sushma , Proudfoot Sarah , Schill Rebecca L , Fatema Kaniz , Thomas Michael J , Kallinger Rachel , Civelek Mete , Haeusler Rebecca A , Remaley Alan T , Neufeld Edward B , Malide Daniela A , Sahoo Daisy , Sorci-Thomas Mary G
Published: March 2019

Adipose tissue undergoes distinct structural remodeling in response to increases in fat mass, as in obesity. During this process, the extracellular matrix (ECM) plays a pivotal role with respect to both adipocyte receptor signaling and structural remodeling. One ECM protein, procollagen endopeptidase enhancer 2 (PCPE2), has been shown by our lab to work in partnership with the scavenger receptor class B type I (SR-BI) to regulate adipocyte cholesterol homeostasis. To investigate the functional impact of the partnership between SR-BI and PCPE2, we studied mature adipocytes differentiated from murine embryonic fibroblasts (MEF) and compared to our adipose specific PCPE2 knockout mice. Our studies showed the presence of an SR-BI-PCPE2 protein complex based on the co-immunoprecipitation of PCPE2 with SR-BI in both differentiated MEFs and mouse adipose tissue. Furthermore, stimulated emission depletion (STED) microscopy showed SR-BI and PCPE2 localized on the lipid droplet surface separated by distances ranging from 24 – 90 nm, suggesting that the SR-BI-PCPE2 complex may contain other proteins. Most significantly, a loss of SR-BI function was shown in PCPE2-deficient adipocytes by the SR-BI mediated HDL binding and cholesteryl ester (CE) uptake assays. Compared to differentiated MEFs from

Ldlr
-/-

mice, binding of HDL to differentiated MEFs from

Ldlr
-/-
Pcpe2
-/-

mice was reduced by 75%, and HDL cholesteryl oleyl ether uptake was reduced by 50%. Disruption of adipocyte SR-BI function in the absence of PCPE2 also reduced free cholesterol efflux indicating substantial disruption to intracellular cholesterol homeostasis. Moreover, analysis of RNAseq from several human clinical studies show that PCPE2 mRNA expression is positively correlated with fat mass in specific adipose depots, while negatively correlated with plasma insulin, glucose and triglyceride levels, with the latter two more robust in type 2 diabetic populations (FDR < 0.004). Overall, these results suggest that the partnership between SR-BI and PCPE2 in adipocytes plays an important role in cholesterol trafficking and homeostasis which influences fat mass expansion, glucose tolerance and insulin resistance.
Abstract 047: The Signaling Adapter Tumor-Necrosis Receptor Associated Factor 1 (TRAF-1) Regulates Thrombosis and Haemostasis in Mice
Authors: Anto Michel Nathaly , Bode Christoph , Zirlik Andreas , Wolf Dennis
Published: March 2019

Background:
The signaling pathways linking inflammatory and thrombotic circuits in platelets are only poorly understood. Here, we tested the role of the inflammatory signaling adapter TRAF-1, which bundles TNF-, TLR, and IL1-signaling, in platelets.

Methods and Results:
To establish a role for platelet expressed TRAF-1, we verified its expression in
in vitro
generated mouse thrombi in immunohistochemistry and western blot. Blood clots generated from blood of
Traf1
-deficient mice were smaller, suggestive of a defective plasmatic coagulation. In accord, tail bleeding time was increased by ~4-fold in
Traf1
-/-
mice. Genetically chimeric mice generated by bone marrow transplantations with a selective deficiency of
Traf1
in bone-marrow-derived leukocytes showed no changes in bleeding time, suggesting that the prolonged bleeding time in
Traf1
-/-
mice was regulated by vascular/stromal cells. In a gene expression array of
Traf1
-/-
endothelial cells, several factors that regulate coagulation, including fibrinogen, tumor-homing peptide (F3), and Von Willebrand factor (vWF) were reduced. In addition to this vascular phenotype, expression of P-selectin and the activation epitope Jon/A induced by
in vitro
ADP and thrombin stimulation was reduced in TRAF-1-deficient platelets. As a consequence,
in vivo
thrombus-generation in the mesenterium was delayed with an enhanced rate of emboli in
Traf1
-/-
mice – an effect that was confirmed in mice transferred with
TRAF1
-/-
platelets, and in mice with a selective deficiency of
Traf1
in bone-marrow-derived cells. Finally, we demonstrate TRAF-1 protein expression in human coronary thrombi and the presence of
TRAF1
-transcripts in RNA-sequencing of human platelets.
TRAF1
-mRNA expression was down-regulated in collagen and TRAP stimulated human platelets and correlated with the gene expression of several upstream platelet-receptors, including
EDA2R
,
RELT
, and
CD137.

Conclusion:
We present the novel finding that the pro-inflammatory signaling adapter TRAF-1 is expressed in mouse and human thrombi and participates in coagulation and thrombosis by vascular and platelet-mediated pathways. These findings emphasize the connection of inflammatory signaling and haemostasis.
Abstract 049: Novel Tirofiban Conjugate for the
in vivo
Detection of Activated Platelets
Authors: Ha Khanh Q , Zheng Xiaoxin , Kessinger Chase , Jaffer Farouc , McCarthy Jason
Published: March 2019

Background:
Current intracoronary imaging approaches such as intravascular ultrasound and optical coherence tomographic imaging do not specifically detect activated platelets, a key cell that drives stent thrombosis, a life-threatening condition following percutaneous coronary intervention (PCI). Glycoprotein IIb/IIIa (GPIIb/IIIa) is the key receptor involved in platelet activation and is a validated target for both therapeutic approaches and diagnostic imaging. A GPIIb/IIIa-targeted fluorescent imaging agent, used in conjunction with a catheter-based NIR fluorescence imaging system may provide powerful tool to detect thrombus prone stents.

Methods:
A NIRF conjugate of tirofiban (Tf), a clinical IIb/IIIa antagonist, was synthesized via the chemical modification of the sulfonamide fragment of the molecule with a benzoic acid moiety and subsequent addition of the PEG-modified fluorophore, CyAl5.5 (ex/em 675/695nm). The efficacy of the conjugate was validated
in vivo
in the FeCl
3
-induced model of femoral thrombosis, and compared to the free dye or blocking with the parent drug Tf. After injection, serial intravital fluorescence microscopy (IVFM) was carried out for one hour, followed by histological examination of binding.

Results:
A NIR fluorescent conjugate of Tf was successfully synthesized via an optimized multi-step synthesis, with the identity and purity of the agent fully characterized after the final HPLC-based purification. IVFM characterization of conjugate efficacy showed an increase in NIRF signal in the thrombus over the initial 30 minutes, followed by a slow decrease until the endpoint of the experiment (1 h). The NIRF signal was 92% ablated by preinjection of excess Tf. Free dye did not display any localization to the thrombus. Histological analysis showed significant accumulation within the thrombus that co-localized with GPIIb/IIIa expression.

Conclusions:
A novel NIR fluorescent Tf conjugate has been developed that specifically binds to GPIIb/IIIa, and could be used for the detection of activated platelets in experimental thrombi
in vivo
. This novel agent should permit assessment of GPIIb/IIIa activation in a broad range of biological processes and may also aid in the detection of thrombosis-prone stents.
Abstract 100: High-throughput Screening of FDA-approved Drugs Identifies Novel Inhibitors of Macropinocytosis
Authors: Lin Hui-Ping , Ghoshal Pushpankur , Singla Bhupesh , Faulkner Jessica L , Shaw Mary C , O’Connor Paul M , She Jin-Xiong , Belin de Chantemele Eric J , Csányi Gábor
Published: March 2019

Aims:
Macropinocytosis has been implicated in atherosclerosis, cancer, allergic disorders, and other pathologies. Unfortunately, most currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. The goal of the present study was to perform a high-throughput screen (HTS) of FDA-approved drugs to identify new, clinically relevant inhibitors of macropinocytosis.

Methods:
In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC
50
values, and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis, clathrin- and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to investigate the mechanisms by which selected compounds inhibit macropinocytosis.

Results:
The HTS campaign identified 14 compounds that at ~10 μM concentration inhibit >95% of macropinocytotic solute internalization. Our results demonstrated that three lead compounds, namely imipramine, phenoxybenzamine, and vinblastine, potently inhibit (IC
50
≤ 130 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Mechanistically, we found that imipramine inhibits translocation of ADP-ribosylation factor 6 to the plasma membrane and prevents membrane ruffle formation, a critical early step leading to macropinocytosis. Imipramine inhibited macropinocytosis in multiple cell types, including cancer cells, dendritic cells, and macrophages. Finally, incubation of macrophages with imipramine inhibited nLDL macropinocytosis and foam cell formation
in vitro
.

Innovation and Conclusion:
The identified macropinocytosis inhibitors may prove useful as new pharmacological tools to more fully discern the role of macropinocytosis in pathological processes and as therapeutic agents in various disorders involving macropinocytosis.
Abstract 123: Establishing a Zebrafish Model of Ischemic Stroke Induced by Photochemical Thrombosis: a Novel Platform for Translational Researches
Authors: Lee I-Ju , Yang Ya-Qi , Chang Wei-Tien , Liau Ian
Published: March 2019

Introduction:
Ischemic stroke is a leading cause of death and disability worldwide. Animal models of stroke are important for translational researches. We herein developed a novel zebrafish model of ischemic stroke using selective photochemical thrombosis, based on which further pathophysiological studies and pharmacological screening can be done.

Method:
Using 5 day post-fertilization zebrafish larva, we injected a photosensitizer rose bengal (120 pg) into the cardinal vein and employed a green laser (λ = 532 nm) targeting at selected cerebral arteries to induce photochemical thrombosis. The fluorescent FITC-dextran was used to stain the larval blood. The dynamic cerebral blood flow was continuously monitored. The neuronal death 12 h after arterial occlusion was indicated by acridine orange (AO). A neurological scoring system measuring the movement, coordination, and response to stimuli was established to determine the neurological function at 24 h. Thrombolysis using recombinant tissue plasminogen activator (rt-PA, 300 ng) at 0.5 and 3 h post-occlusion with comparison of the above parameters was used to demonstrate the potential application of this model.

Results:
Photochemical thrombosis consistently induced occlusion of the selected cerebral arteries. Occlusion at the basilar artery led to high mortality (73.3% at 12 h). All zebrafish survived the occlusion at the 1
st
branch of central artery (CtA), but showed significantly increased AO stain and worsened neurological scores than control. Early treatment of rt-PA at 0.5 h post-occlusion resulted in a higher re-canalization rate than that at 3 h (40.0 vs. 26.7%). Moreover, the early treatment group exhibited significantly lower AO stain (42.2 ± 9.4 vs. 102.6 ± 17.4 a.u.,
P
< 0.05) and improved neurological scores (4.2 ± 0.8 vs. 2.7 ± 1.1,
P
< 0.05). In contrast, no significant improvement was shown in the delayed treatment group even if the occluded artery had been reopened.

Conclusion:
This zebrafish model of ischemic stroke demonstrates characteristic pathophysiological responses and therapeutic effects seen in clinical stroke. With zebrafish being an increasingly employed model animal, there exist great potentials for this model to be applied in basic researches and translational studies.
Abstract 133: Heterozygous Deletion of Transferrin Receptor 1 Suppresses Angiogenesis in a Mouse Model of Hind Limb Ischemia
Authors: Okuno Keisuke , Naito Yoshiro , Yasumura Seiki , Sawada Hisashi , Nishimura Koichi , Asakura Masanori , Ishihara Masaharu , Masuyama Tohru
Published: March 2019

Objective:
Angiogenesis can be triggered under conditions of ischemia and plays a protective role in peripheral artery disease. Iron is an essential trace mineral in the living body whose intracellular metabolism is regulated by transferrin receptor 1 (TfR1). Excess iron causes tissue damage and has been reported in patients with peripheral artery disease. Therefore, we hypothesized that TfR1 contributes to the pathophysiology of hind limb ischemia through iron overload. The aim of this study is to examine whether TfR1 deletion effects angiogenesis in hind limb ischemia.

Methods and Results:
Since homozygous TfR1 deletion is embryonically lethal, mice with heterozygous TfR1 deletion and their wild type littermates were used in this study. To induce hind limb ischemia, the left femoral artery in 8 - 9 week-old male mice was ligated and stripped. The non-ligated right hind limb was used as a control. Blood flow was measured by laser Doppler blood flowmetry at both a baseline of 1 hour and 28 days after the surgery. In wild type, the ratio of ischemc/non-ischemic hind limb blood flow was significantly decreased to 12% at baseline, and recovered to 62% at day 28. On the other hand, TfR1 deleted mice also showed similar reduction of blood flow at baseline, whereas blood flow recovery at day 28 was attenuated to 42%. Subsequently, both right and left adductor muscles were harvested for Western blot and immunohistochemistry. TfR1 expression in the adductor muscle was decreased in TfR1 deleted mice compared to wild type, regardless of the ligation. Ferritin, a marker for iron storage, was 10 times higher in ischemic muscles than in wild type non-ischemic muscles. In contrast, ferritin levels in TfR1 deleted mice were not altered by the surgery. Finally, immunohistochemistry for CD31 was performed to evaluate angiogenesis. The ischemic adductor muscle of TfR1 deleted mice had few CD31 positive vascular structures.

Conclusions:
Heterozygous deletion of TfR1 attenuated iron overload and angiogenesis in a mouse model of hind limb ischemia. TfR1 may be a novel therapeutic target for the treatment of peripheral artery disease.
Abstract 141: The Role of Epsin in Regulating Lymphangiogenesis in Diabetes
Authors: Chen Hong
Published: March 2019

Lymphangiogenesis occurs in adult tissues of chronic complex diseases, including diabetes. Whether lymphangiogenesis is altered owing to diabetic complication remains unknown. VEGF-C/VEGFR-3 signaling axis fuels the growth of lymphatic vessels. How VEGF-C/VEGFR3 signaling is regulated under pathological conditions, such as diabetes, however, is poorly understood. Here, we report that mice with inducible lymphatic endothelial cell-specific deficiency of epsins 1 and 2 (LEC-iDKO) display enhanced lymphangiogenesis compared to wild type (WT) in diabetic condition. A profound increase in VEGF-C-induced lymphatic vessel growth is exhibited in corneas and subcutaneous Matrigel implanted in diabetic LEC-iDKO mice compared to diabetic WT mice. Conversely, lymphatic endothelial cells isolated from diabetic WT mice show marked impairment in proliferation, migration, and tube formation in response to VEGF-C relative to those from diabetic LEC-iDKO
in vitro
. Mechanistically, reactive oxygen species (ROS) generated from hyperglycemia induces c-Src-dependent VEGFR3 phosphorylation independent of VEGF-C, which parallels c-Src-dependent epsin upregulation through the transcription factor AP-1. Heightened epsin expression induced by ROS facilitates epsin binding to VEGFR3 within Golgi compartment, promoting degradation of newly synthesized VEGFR3, and progressively reducing availability of VEGFR3 at the cell surface. Consequentially, lymphatic-specific epsin loss strengthens insufficient lymphangiogenesis and improves the resolution of tail edema in diabetic mice. Collectively, our data indicate that inhibiting epsin expression protects VEGFR3 against degradation and ameliorates diabetes-triggered downregulation of lymphangiogenesis, providing a plausible original therapeutic strategy to treat lymphatic disease in diabetic patients.
Abstract 146: Performance of a Chromogenic Thrombin Generation Assay in the Thrombophilia Screening in 597 Unselected Patients with a History of VenousThromboembolism
Authors: Toulon Pierre , Martin-Toutain Isabelle , Ankri Annick
Published: March 2019

The HemosIL ThromboPath assay (Instrumentation Laboratory) is a chromogenic assay designed to globally evaluate the functionality of the protein C (PC) pathway. It is based on the ability of endogenous APC generated after activation of PC by a snake venom extract (Protac) to reduce the thrombin generation induced by a reagent containing tissue factor. Briefly, optical density is measured after addition of a thrombin-specific chromogenic substrate in the presence (OD A) or absence (OD B) of Protac. Test results are expressed as the Protac-Induced Coagulation Inhibition percentage (PICI%) that corresponds to the ratio [OD B-OD A]/OD B x 100. A normal test result corresponds to a PICI% above a cut-off level defined as the mean-1 SD of the values measured in 30 healthy controls. To determine the performance of that assay, we retrospectively tested frozen plasma samples from 597 consecutive patients referred for screening of biological risk factors for venous thrombosis (209 M and 388 F, mean age=45.6 years, range 15-100). None was on vitamin K-antagonist or had evidence of liver failure. PICI% was significantly lower in patients who presented with than in those without any PC pathway abnormality [median=69.0% (range: 15.3-96.8), n=101 vs. 89.8 (range: 38.5-98.0), n=496; p<0.0001]. All carriers of the Factor V Leiden mutation (32 heterozygotes, and 1 homozygote) had a PICI% below the cut-off level (88.5%). The same applied to all patients with either a PC deficiency (n=14) or a lupus anticoagulant (n=8). The test sensitivity to congenital and acquired PS deficiency was 97.7% (n=45/46). 41.5% of the patients without abnormality of the PC pathway had a decreased test result (n=206/496). The overall test sensitivity to tested thrombophilia was 99.0% (95%CI=94.6-100), its specificity 58.5% (95%CI=54.0-62.9), its negative predictive value (NPV) 99.7% (95%CI=98.1-100) and its PPV 32.7% (95%CI=27.4-38.2).

The high sensitivity of the HemosIL ThromboPath assay to PC pathway abnormalities together with a high NPV, closed to 100%, associated with a normal test result, suggest the potential interest of that assay as part of the screening strategy of PC pathway abnormalities. In that connection, the economic impact of its introduction is currently evaluated.