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1.
Arterioscler Thromb Vasc Biol ; 41(11): 2740-2755, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34615372

RESUMO

Objective: MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. Approach and Results: We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. Conclusions: These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.


Assuntos
Aorta Torácica/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Adesão Celular , Migração e Rolagem de Leucócitos , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/metabolismo , Monócitos/metabolismo , Receptores de Mineralocorticoides/metabolismo , Adulto , Animais , Aorta Torácica/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Aterosclerose/genética , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Adesão Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Hipoglicemia/tratamento farmacológico , Hipoglicemia/genética , Hipoglicemia/metabolismo , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Macrófagos Peritoneais/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Antagonistas de Receptores de Mineralocorticoides/uso terapêutico , Monócitos/efeitos dos fármacos , Monócitos/patologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Receptores de Mineralocorticoides/efeitos dos fármacos , Receptores de Mineralocorticoides/genética , Fatores Sexuais , Transdução de Sinais , Espironolactona/uso terapêutico , Transcrição Genética , Migração Transendotelial e Transepitelial , Resultado do Tratamento , Células U937 , Adulto Jovem
2.
Front Immunol ; 12: 702345, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34489950

RESUMO

ß2 integrins mediate key processes during leukocyte trafficking. Upon leukocyte activation, the structurally bent ß2 integrins change their conformation towards an extended, intermediate and eventually high affinity conformation, which mediate slow leukocyte rolling and firm arrest, respectively. Translocation of talin1 to integrin adhesion sites by interactions with the small GTPase Rap1 and the Rap1 effector Riam precede these processes. Using Rap1 binding mutant talin1 and Riam deficient mice we show a strong Riam-dependent T cell homing process to lymph nodes in adoptive transfer experiments and by intravital microscopy. Moreover, neutrophils from compound mutant mice exhibit strongly increased rolling velocities to inflamed cremaster muscle venules compared to single mutants. Using Hoxb8 cell derived neutrophils generated from the mutant mouse strains, we show that both pathways regulate leukocyte rolling and adhesion synergistically by inducing conformational changes of the ß2 integrin ectodomain. Importantly, a simultaneous loss of both pathways results in a rolling phenotype similar to talin1 deficient neutrophils suggesting that ß2 integrin regulation primarily occurs via these two pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD18/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Proteínas de Membrana/metabolismo , Talina/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Camundongos , Camundongos Knockout
3.
BMC Cardiovasc Disord ; 21(1): 395, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34399692

RESUMO

BACKGROUND: Knowledge of stenosis in coronary arteries requires an understanding of the cellular and molecular processes that occur throughout the leukocyte rolling process. In this study, the roles of miR-125a-5p and miR-495-3p were investigated on the adhesion of endothelial cells (ECs) isolated from the human aorta. METHODS: Human primary endothelial cells were obtained from the aorta of people who had died of brain death. Whole blood was used to isolate the monocytes. The miR-125 and miR-495 were predicted and transfected into ECs using Poly Ethylene Imine (PEI). The expression levels of adhesion molecules and monocyte recruitment were identified by the RT-qPCR technique and Leukocyte-Endothelial Adhesion Assay kit, respectively. RESULTS: The ICAM-1, ICAM-2 and VCAM-1 expression levels decreased significantly in the miR-495/PEI-transfected ECs (P < 0.05) while in the miR-125/PEI-transfected ECs only the ICAM-2 and ITGB-2 expression levels decreased significantly (P < 0.05) as compared to the miR-synthetic/PEI-transfected ECs. Furthermore, the monocyte adhesion was decreased in the miR-125 and miR-mix/PEI-transfected ECs as compared to the miR-synthetic/PEI-transfected ECs (P = 0.01 and P = 0.04, respectively). CONCLUSION: According to the findings, the efficient relations between miR-125 and adhesion molecules may be responsible for the inhibition of monocyte rolling.


Assuntos
Aorta/metabolismo , Moléculas de Adesão Celular/metabolismo , Adesão Celular , Células Endoteliais/metabolismo , MicroRNAs/metabolismo , Monócitos/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Aorta/citologia , Antígenos CD18/genética , Antígenos CD18/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Iminas/química , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Migração e Rolagem de Leucócitos , MicroRNAs/genética , Polietilenos/química , Transdução de Sinais , Transfecção , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
4.
Commun Biol ; 4(1): 832, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215830

RESUMO

Sialyl-Lewis x (sLex, CD15s) is a tetra-saccharide on the surface of leukocytes required for E-selectin-mediated rolling, a prerequisite for leukocytes to migrate out of the blood vessels. Here we show using flow cytometry that sLex expression on basophils and mast cell progenitors depends on fucosyltransferase 6 (FUT6). Using genetic association data analysis and qPCR, the cell type-specific defect was associated with single nucleotide polymorphisms (SNPs) in the FUT6 gene region (tagged by rs17855739 and rs778798), affecting coding sequence and/or expression level of the mRNA. Heterozygous individuals with one functional FUT6 gene harbor a mixed population of sLex+ and sLex- basophils, a phenomenon caused by random monoallelic expression (RME). Microfluidic assay demonstrated FUT6-deficient basophils rolling on E-selectin is severely impaired. FUT6 null alleles carriers exhibit elevated blood basophil counts and a reduced itch sensitivity against insect bites. FUT6-deficiency thus dampens the basophil-mediated allergic response in the periphery, evident also in lower IgE titers and reduced eosinophil counts.


Assuntos
Basófilos/metabolismo , Fucosiltransferases/genética , Expressão Gênica , Antígeno Sialil Lewis X/biossíntese , Sequência de Bases , Basófilos/citologia , Células Cultivadas , Estudos de Coortes , Selectina E/metabolismo , Fucosiltransferases/deficiência , Perfilação da Expressão Gênica/métodos , Humanos , Contagem de Leucócitos , Migração e Rolagem de Leucócitos/genética , Migração e Rolagem de Leucócitos/fisiologia , Polimorfismo de Nucleotídeo Único , Homologia de Sequência do Ácido Nucleico
5.
Front Immunol ; 12: 687711, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34140956

RESUMO

Leukocyte recruitment to the site of injury is a crucial event in the regulation of an inflammatory response. Tight regulation of interactions between the endothelium and circulating leukocytes is necessary to ensure a protective response to injury does not result in inflammatory disease. Rising interest in the broad immunoregulatory roles displayed by members of the glycan-binding galectin family suggests that these proteins could be an attractive target for therapeutic intervention, since their expression is significantly altered in disease. The focus of this review is to summarize current knowledge on the role of galectins in leukocyte trafficking during inflammation and the clinical approaches being taken to target these interactions for treatment of inflammatory disease.


Assuntos
Adesão Celular , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Galectinas/metabolismo , Inflamação/metabolismo , Migração e Rolagem de Leucócitos , Leucócitos/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Galectinas/antagonistas & inibidores , Humanos , Inflamação/tratamento farmacológico , Inflamação/imunologia , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Transdução de Sinais
6.
Front Immunol ; 12: 663886, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995392

RESUMO

Neutrophils are essential to protect the host against invading pathogens but can promote disease progression in sickle cell disease (SCD) by becoming adherent to inflamed microvascular networks in peripheral tissue throughout the body. During the inflammatory response, leukocytes extravasate from the bloodstream using selectin adhesion molecules and migrate to sites of tissue insult through activation of integrins that are essential for combating pathogens. However, during vaso-occlusion associated with SCD, neutrophils are activated during tethering and rolling on selectins upregulated on activated endothelium that line blood vessels. Recently, we reported that recognition of sLex on L-selectin by E-selectin during neutrophil rolling initiates shear force resistant catch-bonds that facilitate tethering to endothelium and activation of integrin bond clusters that anchor cells to the vessel wall. Evidence indicates that blocking this important signaling cascade prevents the congestion and ischemia in microvasculature that occurs from neutrophil capture of sickled red blood cells, which are normally deformable ellipses that flow easily through small blood vessels. Two recently completed clinical trials of therapies targeting selectins and their effect on neutrophil activation in small blood vessels reveal the importance of mechanoregulation that in health is an immune adaption facilitating rapid and proportional leukocyte adhesion, while sustaining tissue perfusion. We provide a timely perspective on the mechanism underlying vaso-occlusive crisis (VOC) with a focus on new drugs that target selectin mediated integrin adhesive bond formation.


Assuntos
Anemia Falciforme/complicações , Adesão Celular/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/etiologia , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Plaquetas/metabolismo , Moléculas de Adesão Celular/metabolismo , Gerenciamento Clínico , Suscetibilidade a Doenças , Endotélio/metabolismo , Humanos , Migração e Rolagem de Leucócitos/genética , Migração e Rolagem de Leucócitos/imunologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Selectinas/metabolismo
7.
Biophys J ; 120(12): 2511-2520, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-33932434

RESUMO

Leukocyte rolling adhesion, facilitated by selectin-mediated interactions, is a highly dynamic process in which cells roll along the endothelial surface of blood vessel walls to reach the site of infection. The most common approach to investigate cell-substrate adhesion is to analyze the cell rolling velocity in response to shear stress changes. It is assumed that changes in rolling velocity indicate changes in adhesion strength. In general, cell rolling velocity is studied at the population level as an average velocity corresponding to given shear stress. However, no statistical investigation has been performed on the instantaneous velocity distribution. In this study, we first developed a method to remove systematic noise and revealed the true velocity distribution to exhibit a log-normal profile. We then demonstrated that the log-normal distribution describes the instantaneous velocity at both the population and single-cell levels across the physiological flow rates. The log-normal parameters capture the cell motion more accurately than the mean and median velocities, which are prone to systematic error. Lastly, we connected the velocity distribution to the molecular adhesion force distribution and showed that the slip-bond regime of the catch-slip behavior of the P-selectin/PSGL-1 interaction is responsible for the variation of cell velocity.


Assuntos
Selectina L , Selectina-P , Adesão Celular , Migração e Rolagem de Leucócitos , Neutrófilos , Estresse Mecânico
8.
Biochem Soc Trans ; 49(2): 693-704, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33843967

RESUMO

Leukocytes continuously circulate our body through the blood and lymphatic vessels. To survey invaders or abnormalities and defend our body against them, blood-circulating leukocytes migrate from the blood vessels into the interstitial tissue space (leukocyte extravasation) and exit the interstitial tissue space through draining lymphatic vessels (leukocyte intravasation). In the process of leukocyte trafficking, leukocytes recognize and respond to multiple biophysical and biochemical cues in these vascular microenvironments to determine adequate migration and adhesion pathways. As leukocyte trafficking is an essential part of the immune system and is involved in numerous immune diseases and related immunotherapies, researchers have attempted to identify the key biophysical and biochemical factors that might be responsible for leukocyte migration, adhesion, and trafficking. Although intravital live imaging of in vivo animal models has been remarkably advanced and utilized, bioengineered in vitro models that recapitulate complicated in vivo vascular structure and microenvironments are needed to better understand leukocyte trafficking since these in vitro models better allow for spatiotemporal analyses of leukocyte behaviors, decoupling of interdependent biological factors, better controlling of experimental parameters, reproducible experiments, and quantitative cellular analyses. This review discusses bioengineered in vitro model systems that are developed to study leukocyte interactions with complex microenvironments of blood and lymphatic vessels. This review focuses on the emerging concepts and methods in generating relevant biophysical and biochemical cues. Finally, the review concludes with expert perspectives on the future research directions for investigating leukocyte and vascular biology using the in vitro models.


Assuntos
Bioengenharia/métodos , Adesão Celular/fisiologia , Endotélio Vascular/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Leucócitos/metabolismo , Modelos Biológicos , Animais , Membrana Basal/metabolismo , Movimento Celular/fisiologia , Humanos
9.
Biophys J ; 120(11): 2102-2111, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33838138

RESUMO

Many biologically important cell binding processes, such as the rolling of leukocytes in the vasculature, are multivalent, being mediated by large numbers of weak binding ligands. Quantitative agreement between experiments and models of rolling has been elusive and often limited by the poor understanding of the binding and unbinding kinetics of the ligands involved. Here, we present a cell-free experimental model for such rolling, consisting of polymer microspheres whose adhesion to a glass surface is mediated by ligands with well-understood force-dependent binding free energy-short complementary DNA strands. We observe robust rolling activity for certain values of the shear rate and the grafted DNA strands' binding free energy and force sensitivity. The simulation framework developed to model leukocyte rolling, adhesive dynamics, quantitatively captures the mean rolling velocity and lateral diffusivity of the experimental particles using known values of the experimental parameters. Moreover, our model captures the velocity variations seen within the trajectories of single particles. Particle-to-particle variations can be attributed to small, plausible differences in particle characteristics. Overall, our findings confirm that state-of-the-art adhesive dynamics simulations are able to capture the complex physics of particle rolling, boding well for their extension to modeling more complex systems of rolling cells.


Assuntos
Adesivos , Migração e Rolagem de Leucócitos , Adesão Celular , DNA , Leucócitos , Microesferas
11.
Arterioscler Thromb Vasc Biol ; 41(4): 1309-1318, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33626909

RESUMO

[Figure: see text].


Assuntos
Anticorpos Neutralizantes/farmacologia , Aterosclerose/prevenção & controle , Moléculas de Adesão Celular Neuronais/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Proteínas da Matriz Extracelular/antagonistas & inibidores , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Animais , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/metabolismo , Receptor 1 de Quimiocina CX3C/genética , Moléculas de Adesão Celular Neuronais/deficiência , Moléculas de Adesão Celular Neuronais/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/deficiência , Proteínas da Matriz Extracelular/genética , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Masculino , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Placa Aterosclerótica , Receptores de LDL/deficiência , Receptores de LDL/genética , Serina Endopeptidases/deficiência , Serina Endopeptidases/genética , Transdução de Sinais , Células U937
12.
Am J Physiol Heart Circ Physiol ; 320(3): H1185-H1198, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33416452

RESUMO

Ischemia/reperfusion (I/R)-induced rapid inflammation involving activation of leukocyte-endothelial adhesive interactions and leukocyte infiltration into tissues is a major contributor to postischemic tissue injury. However, the molecular mediators involved in this pathological process are not fully known. We have previously reported that caveolin-2 (Cav-2), a protein component of plasma membrane caveolae, regulated leukocyte infiltration in mouse lung carcinoma tumors. The goal of the current study was to examine if Cav-2 plays a role in I/R injury and associated acute leukocyte-mediated inflammation. Using a mouse small intestinal I/R model, we demonstrated that I/R downregulates Cav-2 protein levels in the small bowel. Further study using Cav-2-deficient mice revealed aggravated postischemic tissue injury determined by scoring of villi length in H&E-stained tissue sections, which correlated with increased numbers of MPO-positive tissue-infiltrating leukocytes determined by IHC staining. Intravital microscopic analysis of upstream events relative to leukocyte transmigration and tissue infiltration revealed that leukocyte-endothelial cell adhesive interactions in postcapillary venules, namely leukocyte rolling and adhesion were also enhanced in Cav-2-deficient mice. Mechanistically, Cav-2 deficiency increased plasminogen activator inhibitor-1 (PAI-1) protein levels in the intestinal tissue and a pharmacological inhibition of PAI-1 had overall greater inhibitory effect on both aggravated I/R tissue injury and enhanced leukocyte-endothelial interactions in postcapillary venules in Cav-2-deficient mice. In conclusion, our data suggest that Cav-2 protein alleviates tissue injury in response to I/R by dampening PAI-1 protein levels and thereby reducing leukocyte-endothelial adhesive interactions.NEW & NOTEWORTHY The role of caveolin-2 in regulating ischemia/reperfusion (I/R) tissue injury and the mechanisms underlying its effects are unknown. This study uses caveolin-2-deficient mouse and small intestinal I/R injury models to examine the role of caveolin-2 in the leukocyte-dependent reperfusion injury. We demonstrate for the first time that caveolin-2 plays a protective role from the I/R-induced leukocyte-dependent reperfusion injury by reducing PAI-1 protein levels in intestinal tissue and leukocyte-endothelial adhesive interactions in postcapillary venules.


Assuntos
Caveolina 2/deficiência , Adesão Celular , Células Endoteliais/metabolismo , Doenças do Jejuno/metabolismo , Jejuno/irrigação sanguínea , Migração e Rolagem de Leucócitos , Leucócitos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Migração Transendotelial e Transepitelial , Vênulas/metabolismo , Animais , Caveolina 2/genética , Modelos Animais de Doenças , Células Endoteliais/patologia , Doenças do Jejuno/genética , Doenças do Jejuno/patologia , Leucócitos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Vênulas/patologia
13.
Am J Physiol Heart Circ Physiol ; 320(2): H734-H739, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33337960

RESUMO

The integrin family, an indispensable part of cell-cell and cell-matrix interactions, consists of a group of heterodimeric adhesion receptors formed by α- and ß-integrin subunits. Their wide expression and unique bidirectional signaling pathways allow them to play roles in a variety of biological activities including blood clot formation, cell attachment, and migration. Evidence suggests that integrins are essential regulators of the initiation of acute inflammation, especially two key aspects of this process i.e., vascular permeability and leukocyte recruitment. This mini-review discusses the importance of integrins at the onset of the acute inflammatory response and outlines research advances regarding the function of integrins and their modulators at different stages of this process. Insights into the fine-tuning of integrin signaling during acute inflammation may inspire the design of new drugs for inflammatory diseases.


Assuntos
Antígenos CD18/metabolismo , Permeabilidade Capilar , Quimiotaxia de Leucócito , Endotélio Vascular/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Integrina beta1/metabolismo , Leucócitos/metabolismo , Animais , Adesão Celular , Comunicação Celular , Endotélio Vascular/imunologia , Endotélio Vascular/fisiopatologia , Humanos , Inflamação/imunologia , Inflamação/fisiopatologia , Migração e Rolagem de Leucócitos , Leucócitos/imunologia , Transdução de Sinais , Migração Transendotelial e Transepitelial
14.
Blood ; 137(1): 29-38, 2021 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-32777822

RESUMO

Integrin-mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive, bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin-3 is necessary for leukocyte adhesion; mutations of kindlin-3 cause leukocyte adhesion deficiency type 3. Kindlin-3 binds the ß2-integrin cytoplasmic tail at a site distinct from talin-1, but the molecular mechanism by which kindlin-3 activates ß2-integrins is unknown. In this study, we measured the spatiotemporal dynamics of kindlin-3 and ß2-integrin conformation changes during neutrophil and HL-60 cell rolling and arrest under flow. Using high-resolution quantitative dynamic footprinting microscopy and kindlin-3-fluorescent protein (FP) fusion proteins, we found that kindlin-3 was recruited to the plasma membrane in response to interleukin-8 (IL-8) before induction of the H+ ß2-integrin conformation. Intravital imaging revealed that EGFP-kindlin-3-reconstituted, kindlin-3-knockout neutrophils arrest in vivo in response to CXCL1. EGFP-kindlin-3 in primary mouse neutrophils was also recruited to the plasma membrane before arrest. Upon arrest, we found small clusters of high-affinity ß2-integrin molecules within large areas of membrane-proximal kindlin-3 FP. Deletion of kindlin-3 or its pleckstrin homology (PH) domain in neutrophil-like HL-60 cells completely abolished H+ ß2-integrin induction. IL-8 also triggered recruitment of the isolated kindlin-3 PH domain to the plasma membrane before arrest. In summary, we showed that the kindlin-3 PH domain is necessary for recruitment to the plasma membrane, where full-length kindlin-3 is indispensable for the induction of high-affinity ß2-integrin.


Assuntos
Antígenos CD18/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo , Animais , Membrana Celular/metabolismo , Células HL-60 , Humanos , Camundongos , Transporte Proteico/fisiologia
15.
Cardiovasc Res ; 117(1): 162-177, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32077922

RESUMO

AIMS: Heart failure (HF) ensuing myocardial infarction (MI) is characterized by the initiation of a systemic inflammatory response. We aimed to elucidate the impact of myelomonocytic cells and their activation by angiotensin II on vascular endothelial function in a mouse model of HF after MI. METHODS AND RESULTS: HF was induced in male C57BL/6J mice by permanent ligation of the left anterior descending coronary artery. Compared to sham, HF mice had significantly impaired endothelial function accompanied by enhanced mobilization of Sca-1+c-Kit+ haematopoietic stem cells and Sca-1-c-Kit+ common myeloid and granulocyte-macrophage progenitors in the bone marrow as well as increased vascular infiltration of CD11b+Ly6G-Ly6Chigh monocytes and accumulation of CD11b+ F4/80+ macrophages, assessed by flow cytometry. Using mice with Cre-inducible expression of diphtheria toxin receptor in myeloid cells, we selectively depleted lysozyme M+ myelomonocytic cells for 10 days starting 28 days after MI. While the cardiac phenotype remained unaltered until 38 days post-MI, myeloid cell depletion attenuated vascular accumulation of Nox2+CD45+ cells, endothelial dysfunction, oxidative stress, and vascular expression of adhesion molecules and angiotensin II receptor type 1 (AT1R). Pharmacological blockade of this receptor for 4 weeks did not significantly alter cardiac function, but mimicked the effects of myeloid cell depletion: telmisartan (20 mg/kg/day, fed to C57BL/6J mice) diminished bone marrow myelopoesis and myeloid reactive oxygen species production, attenuated endothelial leucocyte rolling and vascular accumulation of CD11b+Ly6G-Ly6Chigh monocytes and macrophages, resulting in improved vascular function with less abundance of Nox2+CD45+ cells. CONCLUSION: Endothelial dysfunction in HF ensuing MI is mediated by inflammatory Nox2+ myeloid cells infiltrating the vessel wall that can be targeted by AT1R blockade.


Assuntos
Angiotensina II/metabolismo , Células Endoteliais/metabolismo , Insuficiência Cardíaca/etiologia , Células Mieloides/enzimologia , Infarto do Miocárdio/complicações , NADPH Oxidase 2/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Vasculite/etiologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/imunologia , Migração e Rolagem de Leucócitos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Monócitos/metabolismo , Muramidase/genética , Muramidase/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/imunologia , Estresse Oxidativo , Transdução de Sinais , Telmisartan/farmacologia , Vasculite/tratamento farmacológico , Vasculite/enzimologia , Vasculite/imunologia
16.
J Trauma Acute Care Surg ; 90(2): 274-280, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33093292

RESUMO

BACKGROUND: Acute traumatic coagulopathy often accompanies traumatic brain injury (TBI) and may impair cognitive recovery. Antithrombin III (AT-III) reduces the hypercoagulability of TBI. Antithrombin III and heparinoids such as enoxaparin (ENX) demonstrate potent anti-inflammatory activity, reducing organ injury and modulating leukocyte (LEU) activation, independent of their anticoagulant effect. It is unknown what impact AT-III exerts on cerebral LEU activation and blood-brain barrier (BBB) permeability after TBI. We hypothesized that AT-III reduces live microcirculatory LEU-endothelial cell (EC) interactions and leakage at the BBB following TBI. METHODS: CD1 mice (n = 71) underwent either severe TBI (controlled cortical impact (CCI), 6-m/s velocity, 1-mm depth, and 4-mm diameter) or sham craniotomy and then received either AT-III (250 IU/kg), ENX (1.5 mg/kg), or vehicle (saline) every 24 hours. Forty-eight hours post-TBI, cerebral intravital microscopy visualized in vivo penumbral microvascular LEU-EC interactions and microvascular leakage to assess BBB inflammation/permeability. Body weight loss and the Garcia neurological test (motor, sensory, reflex, balance) served as surrogates of clinical recovery. RESULTS: Both AT-III and ENX similarly reduced in vivo penumbral LEU rolling and adhesion (p < 0.05). Antithrombin III also reduced live BBB leakage (p < 0.05). Antithrombin III animals demonstrated the least 48-hour body weight loss (8.4 ± 1%) versus controlled cortical impact and vehicle (11.4 ± 0.5%, p < 0.01). Garcia neurological test scores were similar among groups. CONCLUSION: Antithrombin III reduces post-TBI penumbral LEU-EC interactions in the BBB leading to reduced neuromicrovascular permeability. Antithrombin III further reduced body weight loss compared with no therapy. Further study is needed to determine if these AT-III effects on neuroinflammation affect longer-term neurocognitive recovery after TBI.


Assuntos
Antitrombina III/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Hemorragia Encefálica Traumática/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Animais , Hemorragia Encefálica Traumática/sangue , Ensaios de Migração de Leucócitos , Modelos Animais de Doenças , Enoxaparina/farmacologia , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Masculino , Camundongos
17.
J Ethnopharmacol ; 269: 113697, 2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33316364

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Leaves from Ocimum kilimandscharicum Gürke (Lamiaceae) are popularly used against articular pain. AIM OF STUDY: The aim of this study was to test the anti-inflammatory and anti-hyperalgesic (analgesic) properties of the essential oil and camphor isolated from O. Kilimandscharicum leaves (EOOK) in 4 models including zymosan induced-articular inflammation model in mice. MATERIAL AND METHODS: For in vivo models, EOOK was tested in carrageenan-induced paw edema model with oral doses of 30, 100, and 300 mg/kg (oral administration = p.o.) and in zymosan-induced articular inflammation (including knee edema, leukocyte infiltration, mechanical hyperalgesia and nitric oxide), EOOK (100 mg/kg, p. o.) and camphor (30 mg/kg, p. o.) were tested. EOOK (100 mg/kg, p. o.) was tested in the rolling and also in the adhesion of leukocytes to the mesenteric microcirculation in situ model of carrageenan induced inflammation and EOOK (1, 3, 10, 30, and 60 µg/mL) was tested in vitro against neutrophils chemotaxis induced by N-formyl methionyl leucyl phenylalanine (fMLP). RESULTS: The treatment with EOOK significantly inhibited the carrageenan-induced edema, mechanical and cold hyperalgesia. Both, EOOK and camphor inhibited all articular parameters induced by zymosan. In situ intravitral microscopy analysis, EOOK significantly inhibited carrageenan-induced leukocyte rolling and adhesion. In vitro neutrophils chemotaxis, EOOK inhibited the leukocyte chemotaxis induced by fMLP. CONCLUSIONS: The present study showed that EOOK inhibited pain and inflammatory parameters contributing, at least in part, to explain the popular use of this plant as analgesic natural agent. This study also demonstrates that camphor and some known anti-inflammatory compounds present in EOOK could contribute for analgesic and anti-inflammatory articular properties.


Assuntos
Analgésicos/farmacologia , Anti-Inflamatórios/farmacologia , Artralgia/tratamento farmacológico , Cânfora/farmacologia , Ocimum/química , Óleos Voláteis/farmacologia , Analgésicos/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Artralgia/induzido quimicamente , Cânfora/isolamento & purificação , Cânfora/uso terapêutico , Carragenina/toxicidade , Modelos Animais de Doenças , Edema/induzido quimicamente , Edema/tratamento farmacológico , Hiperalgesia/induzido quimicamente , Hiperalgesia/tratamento farmacológico , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Articulações/efeitos dos fármacos , Traumatismos do Joelho/induzido quimicamente , Traumatismos do Joelho/tratamento farmacológico , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Masculino , Camundongos , Neutrófilos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óleos Voláteis/isolamento & purificação , Óleos Voláteis/uso terapêutico , Folhas de Planta/química , Líquido Sinovial/efeitos dos fármacos , Zimosan/toxicidade
18.
J Immunol ; 205(12): 3300-3310, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33199537

RESUMO

Gout is a painful arthritic inflammatory disease caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a microtubule-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, alleviates the painful inflammatory response to MSU crystals. Using i.p. and intra-articular mouse models of gout-like inflammation, we found that GEF-H1/GEF-H1/AHRGEF2, a microtubule-associated Rho-GEF, was necessary for the inhibitory effect of colchicine on neutrophil recruitment. GEF-H1 was required for neutrophil polarization in response to colchicine, characterized by uropod formation, accumulation of F-actin and myosin L chain at the leading edge, and accumulation of phosphorylated myosin L chain, flotillin-2, and P-selectin glycoprotein ligand-1 (PSGL-1) in the uropod. Wild-type neutrophils that were pre-exposed to colchicine failed to roll or accumulate on activated endothelial monolayers, whereas GEF-H1 knockout (GEF-H1-/-) neutrophils were unaffected by treatment with colchicine. In vivo, colchicine blocked MSU-induced recruitment of neutrophils to the peritoneum and the synovium in wild-type mice, but not in GEF-H1-/- mice. Inhibition of macrophage IL-1ß production by colchicine was independent of GEF-H1, supporting a neutrophil-intrinsic mode of action. Our results suggest that the anti-inflammatory effects of colchicine in acute gout-like inflammation can be accounted for by inhibition of neutrophil-rolling interactions with the inflamed vasculature and occurs through GEF-H1-dependent neutrophil stimulation by colchicine. These results contribute to our understanding of the therapeutic action of colchicine, and could inform the application of this drug in other conditions.


Assuntos
Colchicina/farmacologia , Gota , Migração e Rolagem de Leucócitos , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos , Fatores de Troca de Nucleotídeo Guanina Rho/imunologia , Actinas/genética , Actinas/imunologia , Animais , Modelos Animais de Doenças , Gota/tratamento farmacológico , Gota/genética , Gota/imunologia , Gota/patologia , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Migração e Rolagem de Leucócitos/genética , Migração e Rolagem de Leucócitos/imunologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Cadeias Leves de Miosina , Neutrófilos/imunologia , Neutrófilos/patologia , Fatores de Troca de Nucleotídeo Guanina Rho/genética
19.
Microvasc Res ; 132: 104067, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32877697

RESUMO

Edema formation due to the collapse of physiological barriers and the associated delayed healing process is still a central problem in the treatment of burn injuries. In healthy individuals, tight junctions form a barrier to fluid and small molecules. Cingulin is a cytoplasmic component of tight junctions and is involved in the regulation of the paracellular barrier. Endothelial specific cingulin knock-out mice provide new insight into the influence of tight junction proteins on edema formation and angiogenesis during wound healing. Knock-out mice lacking the head domain of cingulin in endothelial cells (CgnΔEC) were created by breeding Cgnfl/fl mice with Tie1-cre mice. Using a no-touch hot air jet a burn trauma was induced on the ear of the mouse. Over a period of 12 days microcirculatory parameters such as edema formation, angiogenesis and leukocyte-endothelial interactions were visualized using intravital fluorescence microscopy. At baseline, CgnΔEC mice surprisingly showed significantly less tracer extravasation compared to Cgnfl/fl littermates, whereas, after burn injury, edema was consistently higher in CgnΔEC mice. Non-perfused area after wounding was increased, but there was no difference in vessel diameters, contraction or dilation of arteries in CgnΔEC mice. Moreover, cingulin knock-out did not cause a difference in leukocyte adhesion after burn injury. In summary, cingulin limits non-perfused area after burn injury and maintains the paracellular barrier of blood vessels. Since edema formation with serious systemic effects is a central problem of burn wounds, understanding the importance of tight junction proteins might help to find new treatment strategies for burn wounds.


Assuntos
Queimaduras/metabolismo , Edema/metabolismo , Células Endoteliais/metabolismo , Proteínas de Membrana/metabolismo , Microvasos/metabolismo , Pele/irrigação sanguínea , Junções Íntimas/metabolismo , Cicatrização , Animais , Queimaduras/genética , Queimaduras/patologia , Permeabilidade Capilar , Modelos Animais de Doenças , Edema/genética , Edema/patologia , Células Endoteliais/patologia , Migração e Rolagem de Leucócitos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microvasos/patologia , Neovascularização Fisiológica , Transdução de Sinais , Junções Íntimas/genética , Junções Íntimas/patologia
20.
Biosens Bioelectron ; 168: 112558, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32911451

RESUMO

Recruitment of circulating leukocytes to sites of infection is of utmost importance in the development, propagation, and outcome of sepsis. These multi-step processes are mediated by interactions between adhesion receptors of leukocytes and cell adhesion molecules (CAMs) of endothelial cells, such as P-selectin, E-selectin and ICAM-1. However, the potential utility of the CAMs-facilitated leukocyte capture has not been thoroughly investigated as an index of the host response to infection for diagnostic purposes. Here, we report that the systemic infection affects the expression of CAMs ligands on leukocytes, upregulating the expression of P-selectin ligand-1 (PSGL-1) and increasing the number of PSGL-1- and E-selectin ligand-1 (ESL-1)-expressing leukocyte levels in septic blood. We leveraged this finding to determine infection by measuring the increased adhesion of leukocytes to an inflammatory vascular endothelium-mimicking microchannel coated with CAMs. We successfully validated that the proposed method can significantly differentiate infection in bacteremia and endotoxemia models in rats as early as an hour post-infection using a finger-prick volume of blood (50 µL), which were unachievable with the conventional diagnostic methods.


Assuntos
Técnicas Biossensoriais , Migração e Rolagem de Leucócitos , Animais , Adesão Celular , Células Endoteliais , Endotélio Vascular , Dispositivos Lab-On-A-Chip , Leucócitos , Selectina-P , Ratos
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