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1.
Colloids Surf B Biointerfaces ; 207: 112002, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34343911

RESUMO

Various ligand-functionalized liposomes have been developed for targeted therapies. Typically, the binding properties of the ligands and targeted proteins are measured with surface plasmon resonance (SPR), where the proteins are immobilized on a rigid surface. However, the difference of protein-ligand binding kinetics between liposome-conjugated protein and rigid surface-conjugated protein is not fully understood. In this work, the binding kinetics of P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin conjugated on liposome and on rigid surfaces are investigated with Atomic Force Microscopy (AFM). The results suggest that protein orientation and diffusion on liposomal membrane can alter the binding kinetics of the protein-ligand interaction. Specifically, the association and dissociation rate constant of AFM probe-conjugated E-selectin and glass-conjugated PSGL-1 are measured as 9.32 × 104 M-1s-1 and 1.54 s-1, respectively. While for the liposome-conjugated E-selectin and glass-conjugated PSGL-1, the kinetic constants are measured as 5.00 × 107 M-1s-1 and 2.76 s-1, respectively. Thus, there is an order's magnitude increase of binding affinity (from kd = 16.51 µM to kd = 0.06 µM) when protein is attached to liposome compared to attached to a rigid surface. The results might provide better understanding and pave the way for the future design of the ligand-targeted liposomes.


Assuntos
Selectina E , Lipossomos , Selectina E/metabolismo , Cinética , Ligantes , Glicoproteínas de Membrana , Microscopia de Força Atômica , Ligação Proteica
2.
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
3.
Commun Biol ; 4(1): 868, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262131

RESUMO

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.


Assuntos
Selectina E/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Microfluídica/métodos , Imagem Individual de Molécula/métodos , Algoritmos , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Humanos , Leucemia Mieloide Aguda/patologia , Ligantes , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência/métodos , Modelos Biológicos
4.
Nat Commun ; 12(1): 4547, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315900

RESUMO

The human pathogen Neisseria meningitidis can cause meningitis and fatal systemic disease. The bacteria colonize blood vessels and rapidly cause vascular damage, despite a neutrophil-rich inflammatory infiltrate. Here, we use a humanized mouse model to show that vascular colonization leads to the recruitment of neutrophils, which partially reduce bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles, adhesion molecules such as E-Selectin are not expressed on the endothelium, and intravascular neutrophil recruitment is minimal. Our results indicate that the colonization of capillaries and arterioles by N. meningitidis creates an intravascular niche that precludes the action of neutrophils, resulting in immune escape and progression of the infection.


Assuntos
Arteríolas/microbiologia , Derme/irrigação sanguínea , Neisseria meningitidis/crescimento & desenvolvimento , Neutrófilos/microbiologia , Adulto , Animais , Arteríolas/patologia , Aderência Bacteriana , Capilares/microbiologia , Capilares/patologia , Moléculas de Adesão Celular/metabolismo , Contagem de Colônia Microbiana , Selectina E/metabolismo , Endotélio Vascular/microbiologia , Endotélio Vascular/patologia , Feminino , Fímbrias Bacterianas/metabolismo , Xenoenxertos , Humanos , Inflamação/patologia , Masculino , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/patologia , Camundongos SCID , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Fagocitose , Fatores de Tempo , Regulação para Cima , Adulto Jovem
5.
Biomed Pharmacother ; 140: 111765, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34058438

RESUMO

Xenocoumacin (Xcn) 1 and 2 are the major antibiotics produced by the insect-pathogenic bacterium Xenorhabdus nematophila. Although the antimicrobial activity of Xcns has been explored, research regarding their action on mammalian cells is lacking. We aimed to investigate the action of Xcns in the context of inflammation and angiogenesis. We found that Xcns do not impair the viability of primary endothelial cells (ECs). Particularly Xcn2, but not Xcn1, inhibited the pro-inflammatory activation of ECs: Xcn2 diminished the interaction between ECs and leukocytes by downregulating cell adhesion molecule expression and blocked critical steps of the NF-κB activation pathway including the nuclear translocation of NF-κB p65 as well as the activation of inhibitor of κBα (IκBα) and IκB kinase ß (IKKß). Furthermore, the synthesis of pro-inflammatory mediators and enzymes, nitric oxide (NO) production and prostaglandin E2 (PGE2), inducible NO synthase (iNOS), and cyclooxygenase-2 (COX-2), was evaluated in leukocytes. The results showed that Xcns reduced viability, NO release, and iNOS expression in activated macrophages. Beyond these anti-inflammatory properties, Xcn2 effectively hindered pro-angiogenic processes in HUVECs, such as proliferation, undirected and chemotactic migration, sprouting, and network formation. Most importantly, we revealed that Xcn2 inhibits de novo protein synthesis in ECs. Consequently, protein levels of receptors that mediate the inflammatory and angiogenic signaling processes and that have a short half-live are reduced by Xcn2 treatment, thus explaining the observed pharmacological activities. Overall, our research highlights that Xcn2 exhibits significant pharmacological in vitro activity regarding inflammation and angiogenesis, which is worth to be further investigated preclinically.


Assuntos
Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios/farmacologia , Benzopiranos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Camundongos , NF-kappa B/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
6.
Aging (Albany NY) ; 13(8): 11942-11953, 2021 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-33875621

RESUMO

Atherosclerosis is a chronic inflammatory disease known to be mediated by numerous factors, among which endothelial dysfunction plays a critical role. Oscillatory shear stress induces endothelial cells to lose their anti-atherosclerotic properties and downregulates the expression of the innate protective transcription factor, Krüppel-like factor 2 (KLF2), which is typically upregulated in vascular endothelial cells in response to harmful stimuli. Oxidative stress and inflammation impair endothelial function and damage their survival. Oscillatory shear stress also promotes generation of reactive oxygen species and production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby further promoting endothelial dysfunction and formation of atherosclerotic plaque. A major event in the development of atherosclerotic plaque is rolling and adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules including vascular cellular adhesion molecule 1 and endothelial-selectin. Expression of these molecules is also upregulated by oscillatory shear stress. Estrogen has long been recognized as a protective agent against atherosclerosis, but the mechanisms through which estrogen receptors prevent atherogenesis remain unclear. In the present study, we investigated the role of the G-coupled protein estrogen receptor (GPR30) in oscillatory shear stress- induced endothelial dysfunction. We show that agonism of GPR30 by its specific agonist G1 prevented oscillatory shear stress -induced oxidative stress markers and production of inflammatory cytokines and adhesion molecules. As a result, GPR30 activation suppresses monocytes adhesion to endothelial cells. Furthermore, we demonstrate that GPR30 prevents oscillatory shear stress- induced downregulation of KLF2 via ERK5 pathway. These findings suggest that endothelial GPR30 is potential target to suppress oscillatory shear stress mediated atherogenesis.


Assuntos
Aterosclerose/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/metabolismo , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Aterosclerose/patologia , Adesão Celular/genética , Selectina E/metabolismo , Células Endoteliais , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Quinolinas/uso terapêutico , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Estresse Mecânico , Células THP-1 , Molécula 1 de Adesão de Célula Vascular/metabolismo
7.
FASEB J ; 35(5): e21521, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33811691

RESUMO

Transendothelial migration (TEM) of neutrophils under blood flow is critical in the inflammatory cascade. However, the role of endothelial plasticity in this process is not fully understood. Therefore, we used an in vitro model to test the dynamics of human polymorphonuclear neutrophil (PMN) TEM across lipopolysaccharide-treated human umbilical vein endothelial cell (HUVEC) monolayers. Interestingly, shRNA-E-selectin knockdown in HUVECs destabilized endothelial junctional integrity by reducing actin branching and increasing stress fiber at cell-cell junctions. This process is accomplished by downregulating the activation of cortactin and Arp2/3, which in turn alters the adhesive function of VE-cadherin, enhancing PMN transmigration. Meanwhile, redundant P-selectins possess overlapping functions in E-selectin-mediated neutrophil adhesion, and transmigration. These results demonstrate, to our knowledge, for the first time, that E-selectins negatively regulate neutrophil transmigration through alterations in endothelial plasticity. Furthermore, it improves our understanding of the mechanisms underlying actin remodeling, and junctional integrity, in endothelial cells mediating leukocyte TEM.


Assuntos
Movimento Celular , Selectina E/metabolismo , Endotélio Vascular/fisiologia , Junções Intercelulares/fisiologia , Neutrófilos/fisiologia , Migração Transendotelial e Transepitelial , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Células Cultivadas , Selectina E/genética , Endotélio Vascular/citologia , Humanos , Neutrófilos/citologia , Pseudópodes
8.
Eur J Pharmacol ; 890: 173651, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33049301

RESUMO

α-Conidendrin is a lignan isolated from Taxus wallichiana and other species. In the present study, we demonstrated that α-conidendrin inhibited the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) at an IC50 value of 40-60 µM in human lung adenocarcinoma A549 cells. α-Conidendrin decreased ICAM-1 protein and mRNA expression levels at concentrations of 40-100 µM in TNF-α-stimulated A549 cells. The TNF-α-induced mRNA expression of vascular cell adhesion molecule-1, E-selectin, and cyclooxygenase-2 was also reduced by α-conidendrin. In the TNF-α-induced nuclear factor κB (NF-κB) signaling pathway, α-conidendrin did not influence the translocation of the NF-κB subunit RelA from the cytoplasm to the nucleus at concentrations up to 100 µM. A chromatin immunoprecipitation assay revealed that α-conidendrin at 100 µM reduced the binding of RelA to the ICAM-1 promoter in response to a stimulation with TNF-α. Collectively, these results indicated that α-conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter, thereby reducing ICAM-1 transcription.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Lignanas/farmacologia , Neoplasias Pulmonares/metabolismo , Tetra-Hidronaftalenos/farmacologia , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Cromanos/farmacologia , Ciclo-Oxigenase 2/metabolismo , Selectina E/efeitos dos fármacos , Selectina E/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/toxicidade
9.
Phytomedicine ; 80: 153359, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33188983

RESUMO

BACKGROUND: Cardiovascular diseases, obesity, and insulin resistance demonstrate elements of functional impairment of the endothelium. Treatment of endothelial dysfunction with natural products, such as pomegranate, can open new ways in the treatment of cardiovascular diseases. PURPOSE: The present meta-analysis provides information in highlighting the role of pomegranate in endothelial dysfunction. METHODS: Various databases, such as PubMed, Scopus, Web of Science, Cochrane, and Google Scholar, were searched up to July 2020 using relevant keywords. We have selected the studies that investigated the effects of pomegranate on vascular adhesion factors, including intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and interleukin-6 (IL-6). MD with 95% CrI with 100,000 iterations by using Markov chain Monte Carlo code were used. RESULTS: Pooled effect size of articles in human studies indicated that pomegranate juice was not significantly effective on ICAM-1 [MD: -0.42; CrI: (-1.01, 0.17)], VCAM-1 [MD: -0.20; CrI: (-1.95, 1.40)], and E-selectin [MD: -0.21; CrI: (-1.62, 1.21)] compared to the control group. But it can significantly reduce IL-6 [MD: -1.07; CrI: (-1.90, -0.19)]. CONCLUSION: Generally, present study showed that pomegranate juice has no significant effect on vascular adhesion factors, ICAM-1, VCAM-1, and E-selectin, but can reduce IL-6 significantly. Future prospective randomized clinical trials with longer intervention duration are warranted to obtain a precise conclusion.


Assuntos
Selectina E/sangue , Endotélio Vascular/efeitos dos fármacos , Sucos de Frutas e Vegetais , Molécula 1 de Adesão Intercelular/sangue , Interleucina-6/sangue , Romã (Fruta) , Adolescente , Adulto , Idoso , Doenças Cardiovasculares/dietoterapia , Doenças Cardiovasculares/metabolismo , Selectina E/metabolismo , Endotélio Vascular/fisiopatologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Pessoa de Meia-Idade , Romã (Fruta)/química , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/metabolismo , Adulto Jovem
10.
Int J Mol Sci ; 21(21)2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33167483

RESUMO

Metastasis is the main cause of death among colorectal cancer (CRC) patients. E-selectin and its carbohydrate ligands, including sialyl Lewis X (sLeX) antigen, are key players in the binding of circulating tumor cells to the endothelium, which is one of the major events leading to organ invasion. Nevertheless, the identity of the glycoprotein scaffolds presenting these glycans in CRC remains unclear. In this study, we firstly have characterized the glycoengineered cell line SW620 transfected with the fucosyltransferase 6 (FUT6) coding for the α1,3-fucosyltransferase 6 (FUT6), which is the main enzyme responsible for the synthesis of sLeX in CRC. The SW620FUT6 cell line expressed high levels of sLeX antigen and E-selectin ligands. Moreover, it displayed increased migration ability. E-selectin ligand glycoproteins were isolated from the SW620FUT6 cell line, identified by mass spectrometry, and validated by flow cytometry and Western blot (WB). The most prominent E-selectin ligand we identified was the neural cell adhesion molecule L1 (L1CAM). Previous studies have shown association of L1CAM with metastasis in cancer, thus the novel role as E-selectin counter-receptor contributes to understand the molecular mechanism involving L1CAM in metastasis formation.


Assuntos
Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Selectina E/metabolismo , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Adesão Celular/genética , Neoplasias do Colo/genética , Humanos , Imunoprecipitação , Ligantes , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Ligação Proteica/genética , Transfecção , Células Tumorais Cultivadas
11.
J Chem Inf Model ; 60(10): 5153-5161, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-32941021

RESUMO

The loop at the E-selectin binding site displayed open and close conformations observed in crystal structures before and after complexing with sialyl lewis x (sLex), respectively, and these different conformations were less studied and could affect the binding and dissociation of selectin/sLex that are essential for the recruitment of leukocytes and early inflammatory response. Hereby, we studied the roles of different loop conformations by performing molecular dynamics simulations, including adaptive steered MD simulations and energy calculations. Results suggested that the loop in the E-selectin binding site could switch from open to close conformation after the binding of sLex spontaneously, and the close conformation enhanced the binding by making sLex immersed slightly deeper in the binding site. Potential mean force calculations showed that more work was required for sLex to dissociate when the loop was in the close conformation, benefiting the formation of the catch bonds and prolonging the bonding lifetime by having more durable interactions between sLex and the loop residues in the rebinding step. This study provided atomic and dynamic details of the influence of the loop conformations on E-selectin/sLex interactions and further elucidated their mechanisms.


Assuntos
Selectina E , Oligossacarídeos , Sítios de Ligação , Selectina E/metabolismo , Conformação Molecular , Antígeno Sialil Lewis X
12.
Am J Physiol Endocrinol Metab ; 319(5): E893-E903, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32954825

RESUMO

Saturated fatty acid (SFA) induces proinflammatory response through a Toll-like receptor (TLR)-mediated mechanism, which is associated with cardiometabolic diseases such as obesity, insulin resistance, and endothelial dysfunction. Consistent with this notion, TLR2 or TLR4 knockout mice are protected from obesity-induced proinflammatory response and endothelial dysfunction. Although SFA causes endothelial dysfunction through TLR-mediated signaling pathways, the mechanisms underlying SFA-stimulated inflammatory response are not completely understood. To understand the proinflammatory response in vascular endothelial cells in high-lipid conditions, we compared the proinflammatory responses stimulated by palmitic acid (PA) and other canonical TLR agonists [lipopolysaccharide (LPS), Pam3-Cys-Ser-Lys4 (Pam3CSK4), or macrophage-activating lipopeptide-2)] in human aortic endothelial cells. The expression profiles of E-selectin and the signal transduction pathways stimulated by PA were distinct from those stimulated by canonical TLR agonists. Inhibition of long-chain acyl-CoA synthetases (ACSL) by a pharmacological inhibitor or knockdown of ACSL1 blunted the PA-stimulated, but not the LPS- or Pam3CSK4-stimulated proinflammatory responses. Furthermore, triacsin C restored the insulin-stimulated vasodilation, which was impaired by PA. From the results, we concluded that PA stimulates the proinflammatory response in the vascular endothelium through an ACSL1-mediated mechanism, which is distinct from LPS- or Pam3CSK4-stimulated responses. The results suggest that endothelial dysfunction caused by PA may require to undergo intracellular metabolism. This expands the understanding of the mechanisms by which TLRs mediate inflammatory responses in endothelial dysfunction and cardiovascular disease.


Assuntos
Aorta/metabolismo , Coenzima A Ligases/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Ácido Palmítico/farmacologia , Aorta/efeitos dos fármacos , Selectina E/metabolismo , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Humanos , Insulina/farmacologia , Lipopolissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Triazenos/farmacologia
13.
Medicine (Baltimore) ; 99(38): e22241, 2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32957369

RESUMO

BACKGROUND: Quercetin, a major flavonol, wildly exists in plantage, which has been reported to have an anti-apoptosis and anti-inflammation effects on vascular endothelial cells, but its underlying molecular mechanisms remain unclear. OBJECTIVE: The aim of this study was to investigate the mechanisms of how quercetin inhibits tumor necrosis factor alpha (TNF-α) induced human umbilical vein endothelial cells (HUVECs) apoptosis and inflammation. METHODS AND RESULTS: HUVECs were preconditioned with quercetin for 18 hours, and subsequently treated with TNF-α for 6 hours to induce apoptosis. The expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, ß-actin mRNA was then detected by RT-PCR. Flow cytometry was used to estimate the apoptosis rates, and the expression of activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB) was measured by Western blot. TNF-α induced elevated apoptosis rates and upregulation of VCAM-1, ICAM-1, and E-selectin were meaningfully reduced in HUVECs by pretreatment with quercetin. In addition, quercetin also inhibited the activation of AP-1and NF-κB. CONCLUSION: Results indicate that quercetin could suppress TNF-α induced apoptosis and inflammation by blocking NF-κB and AP-1 signaling pathway in HUVECs, which might be one of the underlying mechanisms in treatment of coronary heart disease.


Assuntos
Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Inflamação/prevenção & controle , NF-kappa B/metabolismo , Quercetina/farmacologia , Fator de Transcrição AP-1/metabolismo , Regulação para Baixo , Selectina E/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismo
14.
Int J Mol Sci ; 21(17)2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872308

RESUMO

Aberrant sialylation is frequently found in pancreatic ductal adenocarcinoma (PDA). α2,3-Sialyltransferases (α2,3-STs) ST3GAL3 and ST3GAL4 are overexpressed in PDA tissues and are responsible for increased biosynthesis of sialyl-Lewis (sLe) antigens, which play an important role in metastasis. This study addresses the effect of α2,3-STs knockdown on the migratory and invasive phenotype of PDA cells, and on E-selectin-dependent adhesion. Characterization of the cell sialome, the α2,3-STs and fucosyltransferases involved in the biosynthesis of sLe antigens, using a panel of human PDA cells showed differences in the levels of sialylated determinants and α2,3-STs expression, reflecting their phenotypic heterogeneity. Knockdown of ST3GAL3 and ST3GAL4 in BxPC-3 and Capan-1 cells, which expressed moderate to high levels of sLe antigens and α2,3-STs, led to a significant reduction in sLex and in most cases in sLea, with slight increases in the α2,6-sialic acid content. Moreover, ST3GAL3 and ST3GAL4 downregulation resulted in a significant decrease in cell migration and invasion. Binding and rolling to E-selectin, which represent key steps in metastasis, were also markedly impaired in the α2,3-STs knockdown cells. Our results indicate that inhibition of ST3GAL3 and ST3GAL4 may be a novel strategy to block PDA metastasis, which is one of the reasons for its dismal prognosis.


Assuntos
Selectina E/metabolismo , Neoplasias Pancreáticas/metabolismo , RNA Interferente Pequeno/farmacologia , Sialiltransferases/genética , Linhagem Celular Tumoral , Movimento Celular , Fucosiltransferases/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Sialiltransferases/antagonistas & inibidores
15.
J Cell Mol Med ; 24(21): 12789-12798, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32985079

RESUMO

In this study, a new water and alkaline-soluble polysaccharide (ALP), with an average molecular weight of 6.63 × 104  Da, was successfully purified from the rhizomes of Atractylodes lancea. GC analysis demonstrated that ALP was a kind of glucan. The effect of the ALP on the interaction between E-selectin and sialyl Lewis X (sLex ) was examined in human osteosarcoma U-2 OS cells. It was obvious that the expression of sLex antigen on the surface of U-2 OS cells was visible under fluorescence microscopy. The addition of ALP (0.5, 1 and 2 mg/mL) resulted in a marked inhibition on the adhesion, migration and invasion of U-2 OS cells to human umbilical vein endothelial cells (HUVECs), which was achieved by the decreased sLex expression on U-2 OS cells. Additionally, the induction of apoptosis can be observed in U-2 OS cells following ALP treatment using TUNEL staining and Annexin V-FITC/PI double-staining analysis on flow cytometry. In conclusion, these results indicated that ALP exerted anti-metastatic activity towards osteosarcoma cells via inhibition of sLex /E-selectin binding, which suggested that ALP could be a potent agent for human osteosarcoma intervention.


Assuntos
Atractylodes/química , Selectina E/metabolismo , Osteossarcoma/patologia , Polissacarídeos/farmacologia , Antígeno Sialil Lewis X/metabolismo , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Monossacarídeos/análise , Metástase Neoplásica , Polissacarídeos/isolamento & purificação , Ligação Proteica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
16.
Biochemistry ; 59(39): 3757-3771, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32901486

RESUMO

Recruitment of circulating cells toward target sites is primarily dependent on selectin/ligand adhesive interactions. Glycosyltransferases are involved in the creation of selectin ligands on proteins and lipids. α1,3-Fucosylation is imperative for the creation of selectin ligands, and a number of fucosyltransferases (FTs) can modify terminal lactosamines on cells to create these ligands. One FT, fucosyltransferase VI (FTVI), adds a fucose in an α1,3 configuration to N-acetylglucosamine to generate sialyl Lewis X (sLex) epitopes on proteins of live cells and enhances their ability to bind E-selectin. Although a number of recombinant human FTVIs have been purified, apart from limited commercial enzymes, they were not characterized for their activity on live cells. Here we focused on establishing a robust method for producing FTVI that is active on living cells (hematopoietic cells and mesenchymal stromal cells). To this end, we used two expression systems, Bombyx mori (silkworm) and Pichia pastoris (yeast), to produce significant amounts of N-terminally tagged FTVI and demonstrated that these enzymes have superior activity when compared to currently available commercial enzymes that are produced from various expression systems. Overall, we outline a scheme for obtaining large amounts of highly active FTVI that can be used for the application of FTVI in enhancing the engraftment of cells lacking the sLex epitopes.


Assuntos
Selectina E/metabolismo , Fucosiltransferases/metabolismo , Polissacarídeos/metabolismo , Células-Tronco/metabolismo , Animais , Bombyx/genética , Linhagem Celular , Linhagem Celular Tumoral , Fucosiltransferases/genética , Expressão Gênica , Humanos , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
PLoS Negl Trop Dis ; 14(7): e0007656, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687542

RESUMO

Platelets drive endothelial cell activation in many diseases. However, if this occurs in Plasmodium vivax malaria is unclear. As platelets have been reported to be activated and to play a role in inflammatory response during malaria, we hypothesized that this would correlate with endothelial alterations during acute illness. We performed platelet flow cytometry of PAC-1 and P-selectin. We measured platelet markers (CXCL4, CD40L, P-selectin, Thrombopoietin, IL-11) and endothelial activation markers (ICAM-1, von Willebrand Factor and E-selectin) in plasma with a multiplex-based assay. The values of each mediator were used to generate heatmaps, K-means clustering and Principal Component analysis. In addition, we determined pair-wise Pearson's correlation coefficients to generate correlation networks. Platelet counts were reduced, and mean platelet volume increased in malaria patients. The activation of circulating platelets in flow cytometry did not differ between patients and controls. CD40L levels (Median [IQ]: 517 [406-651] vs. 1029 [732-1267] pg/mL, P = 0.0001) were significantly higher in patients, while P-selectin and CXCL4 showed a nonsignificant trend towards higher levels in patients. The network correlation approach demonstrated the correlation between markers of platelet and endothelial activation, and the heatmaps revealed a distinct pattern of activation in two subsets of P. vivax patients when compared to controls. Although absolute platelet activation was not strong in uncomplicated vivax malaria, markers of platelet activity and production were correlated with higher endothelial cell activation, especially in a specific subset of patients.


Assuntos
Plaquetas/citologia , Malária Vivax/sangue , Adulto , Plaquetas/metabolismo , Ligante de CD40/genética , Ligante de CD40/metabolismo , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-11/genética , Interleucina-11/metabolismo , Malária Vivax/genética , Malária Vivax/metabolismo , Masculino , Selectina-P/genética , Selectina-P/metabolismo , Ativação Plaquetária , Contagem de Plaquetas , Adulto Jovem
18.
Chem Commun (Camb) ; 56(55): 7549-7552, 2020 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-32579622

RESUMO

A facile enzymatic modular assembly strategy for the preparative-scale synthesis of poly-N-acetyllactosamine (poly-LacNAc) glycans with varied lengths and designed sialylation and/or fucosylation patterns is described. These glycans were printed as a microarray to investigate their interactions with a panel of glycan binding proteins (GBPs). Binding affinities revealed that the avidity of GBPs could be largely affected by the length and the patterns of sialylation and fucosylation.


Assuntos
Glicosiltransferases/química , Polissacarídeos/síntese química , Ascomicetos/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Sequência de Carboidratos , Selectina E/metabolismo , Glicosilação , Griffonia/química , Hemaglutininas/metabolismo , Humanos , Lectinas/metabolismo , Maackia/química , Análise em Microsséries , Estrutura Molecular , Lectinas de Plantas/metabolismo , Polissacarídeos/metabolismo
19.
Biomolecules ; 10(5)2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32354017

RESUMO

Peucedanum ostruthium (L.) Koch, commonly known as masterwort, has a longstanding history as herbal remedy in the Alpine region of Austria, where the roots and rhizomes are traditionally used to treat disorders of the gastrointestinal and respiratory tract. Based on a significant NF-κB inhibitory activity of a P. ostruthium extract (PO-E), this study aimed to decipher those constituents contributing to the observed activity using a recently developed biochemometric approach named ELINA (Eliciting Nature's Activities). This -omics tool relies on a deconvolution of the multicomponent mixture, which was employed by generating microfractions with quantitative variances of constituents over several consecutive fractions. Using an optimized and single high-performance counter-current chromatographic (HPCCC) fractionation step 31 microfractions of PO-E were obtained. 1H NMR data and bioactivity data from three in vitro cell-based assays, i.e., an NF-ĸB reporter-gene assay and two NF-κB target-gene assays (addressing the endothelial adhesion molecules E-selectin and VCAM-1) were collected for all microfractions. Applying heterocovariance analyses (HetCA) and statistical total correlation spectroscopy (STOCSY), quantitative variances of 1H NMR signals of neighboring fractions and their bioactivities were correlated. This revealed distinct chemical features crucial for the observed activities. Complemented by LC-MS-CAD data this biochemometric approach differentiated between active and inactive constituents of the complex mixture, which was confirmed by NF-κB reporter-gene testing of the isolates. In this way, four furanocoumarins (imperatorin, ostruthol, saxalin, and 2'-O-acetyloxypeucedanin), one coumarin (ostruthin), and one chromone (peucenin) were identified as NF-κB inhibiting constituents of PO-E contributing to the observed NF-ĸB inhibitory activity. Additionally, this approach also enabled the disclose of synergistic effects of the PO-E metabolites imperatorin and peucenin. In sum, prior to any isolation an early identification of even minor active constituents, e.g. peucenin and saxalin, ELINA enables the targeted isolation of bioactive constituents and, thus, to effectively accelerate the NP-based drug discovery process.


Assuntos
Anti-Inflamatórios/química , Apiaceae/química , Cumarínicos/análise , Extratos Vegetais/química , Anti-Inflamatórios/farmacologia , Cromatografia Líquida , Cumarínicos/química , Cumarínicos/farmacologia , Selectina E/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Espectrometria de Massas , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Espectroscopia de Prótons por Ressonância Magnética , Molécula 1 de Adesão de Célula Vascular/metabolismo
20.
Cells ; 9(5)2020 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-32397494

RESUMO

Microbial proteins have recently been found to have more benefits in clinical disease treatment because of their better-developed strategy and properties than traditional medicine. In this study, we investigated the effectiveness of a truncated peptide synthesized from the C-terminal sequence of pneumolysin, i.e., C70PLY4, in Streptococcus pneumoniae, in treating chronic inflammatory conditions. It has been shown that C70PLY4 significantly blocks the transendothelial migration of neutrophils and attenuates the formation of atherosclerotic plaque and the secretion of soluble forms of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in high-fat-diet/streptozotocin-induced inflammatory rats. The mechanism and the docking simulation analysis further indicated that C70PLY4 might serve as a Toll-like receptor 4 (TLR4) antagonist by competing for the binding site of MD2, an indispensable protein for lipopolysaccharide (LPS)-TLR4 interaction signaling, on the TLR4 structure. Moreover, compared to the full-length PLY, C70PLY4 seems to have no cytotoxicity in human vascular endothelial cells. Our study elucidated a possible therapeutic efficacy of C70PLY4 in reducing chronic inflammatory conditions and clarified the underlying mechanism. Thus, our findings identify a new drug candidate that, by blocking TLR4 activity, could be an effective treatment for patients with chronic inflammatory diseases.


Assuntos
Inflamação/tratamento farmacológico , Proteínas Mutantes/farmacologia , Proteínas Mutantes/uso terapêutico , Streptococcus pneumoniae/metabolismo , Estreptolisinas/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Sítios de Ligação , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Dieta Hiperlipídica , Selectina E/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos , Camundongos , Simulação de Acoplamento Molecular , Proteínas Mutantes/química , NF-kappa B/metabolismo , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Solubilidade , Estreptolisinas/química , Estreptozocina , Receptor 4 Toll-Like/metabolismo , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo
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