RESUMO
The transcription factor nuclear factor κB (NF-κB) is activated by proinflammatory cytokines, such as tumor necrosis factor α (TNF-α) and Toll-like receptor (TLR) ligands. Screening of NPDepo chemical libraries identified porphyrin derivatives as anti-inflammatory compounds that strongly inhibited the up-regulation of intercellular adhesion molecule-1 (ICAM-1) expression induced by TNF-α, interleukin-1α, the TLR3 ligand, and TLR4 ligand in human umbilical vein endothelial cells. In the present study, the mechanisms of action of porphyrin derivatives were further elucidated using human lung adenocarcinoma A549 cells. Porphyrin derivatives, i.e., dimethyl-2,7,12,18-tetramethyl-3,8-di(1-methoxyethyl)-21H,23H-porphine-13,17-dipropionate (1) and pheophorbide a (2), inhibited TNF-α-induced ICAM-1 expression and decreased the TNF-α-induced transcription of ICAM-1, vascular cell adhesion molecule-1, and E-selectin genes. 1 and 2 reduced the expression of the NF-κB subunit RelA protein for 1 h, which was not rescued by the inhibition of proteasome- and lysosome-dependent protein degradation. In addition, 1 and 2 decreased the expression of multiple components of the TNF receptor 1 complex, and this was accompanied by the appearance of their cross-linked forms. As common components of the NF-κB signaling pathway, 1 and 2 also cross-linked the α, ß, and γ subunits of the inhibitor of NF-κB kinase complex and the NF-κB subunits RelA and p50. Cellular protein synthesis was prevented by 2, but not by 1. Therefore, the present results indicate that porphyrin derivative 1 reduced the expression and increased the cross-linked forms of cellular components required for the NF-κB signaling pathway without affecting global protein synthesis.
Assuntos
Molécula 1 de Adesão Intercelular , NF-kappa B , Porfirinas , Transdução de Sinais , Fator de Necrose Tumoral alfa , Humanos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Molécula 1 de Adesão Intercelular/genética , NF-kappa B/metabolismo , Porfirinas/farmacologia , Porfirinas/química , Células A549 , Selectina E/metabolismo , Selectina E/genética , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Increased bone marrow (BM) homing of NK cells is associated with positive outcome in patients with acute myeloid leukemia (AML) treated within adoptive NK cell transfer trials. While most efforts to further improve the efficacy focus on augmenting NK cell persistence and cytotoxicity, few address their ability to home to the tumor. Here, we decipher how AML growth alters the BM niche to impair NK cell infiltration and how insights can be utilized to resolve this issue. We show that AML development gradually impairs the BM homing capacity of infused NK cells, which was tightly linked to loss of SDF-1α in this environment. AML development also triggered up-regulation of E-selectin on BM endothelial cells. Given the poor E-selectin-binding capacity of NK cells, introduction of fucosyltransferase-7 (FUT7) to the NK cells per mRNA transfection resulted in potent E-selectin binding and stronger adhesion to E-selectin+ endothelial cells. Co-introduction of FUT7 and gain-of-function CXCR4 (CXCR4R334X) redirected NK cell homing to the BM of AML-bearing mice nearly to the levels in AML-free mice. This work shows how impaired NK cell homing caused by AML-induced microenvironmental changes can be overcome by genetic engineering. We speculate our insights can help further advance future NK cell immunotherapies.
Assuntos
Quimiocina CXCL12 , Leucemia Mieloide Aguda , Humanos , Animais , Camundongos , Quimiocina CXCL12/metabolismo , Medula Óssea/patologia , Células Endoteliais/metabolismo , Selectina E/genética , Selectina E/metabolismo , Leucemia Mieloide Aguda/patologia , Células Matadoras Naturais/metabolismo , Células da Medula Óssea/metabolismoRESUMO
The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.
Assuntos
Neovascularização Fisiológica , Doença Arterial Periférica , Camundongos , Animais , Selectina E/genética , Camundongos Endogâmicos C57BL , Músculo Esquelético/irrigação sanguínea , Isquemia/genética , Isquemia/terapia , Terapia Genética/métodos , Doença Arterial Periférica/genética , Doença Arterial Periférica/terapia , Membro Posterior/irrigação sanguínea , Desenvolvimento Muscular , Modelos Animais de DoençasRESUMO
PURPOSE: We have previously shown that the TT genotype (rs579459 location of the ABO gene) is significantly associated with circulating levels of e-selectin in patients with suspected obstructive sleep apnea (OSA). We hypothesized that this genotype would be associated with incident cardiovascular disease (CVD). METHODS: Patients with suspected OSA who had a full diagnostic polysomnogram from 2003 to 2011 were recruited; CV events occurring within 8 years of polysomnography were identified by linkage to provincial health databases. Cox proportional hazards models were used to evaluate the incidence of first CV events as a function of the rs579459 genotype. RESULTS: In this targeted study, 408 patients were studied, and 39 incident events were identified. A larger proportion of patients with the TT genotype had an event (31/247; 12.6%) than the CT and CC genotypes (8/161; 5.0%); in univariate analysis, the TT genotype was significantly associated with CV events (HR = 2.53; 95% CI = 1.16-5.51, p = 0.02). After adjustment for age, AHI, sex, smoking, diabetes, statin use, and BMI, the TT genotype remained a significant predictor (HR = 2.35; 95% CI = 1.02-5.42, p = 0.046). No events were found in patients with an absence of both OSA and the TT genotype (N = 30). The effect of the SNP was partially (16.2%) mediated by e-selectin levels. CONCLUSION: This is the first study to examine genetic variants as a risk factor for incident CVD in the context of OSA. Although these results are preliminary and in need of replication, it suggests that genetic markers may become useful in helping to guide precision clinical care.
Assuntos
Doenças Cardiovasculares , Apneia Obstrutiva do Sono , Humanos , Selectina E/genética , Projetos Piloto , Fatores de Risco , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/genética , Polimorfismo Genético , Apneia Obstrutiva do Sono/diagnóstico , Apneia Obstrutiva do Sono/epidemiologia , Apneia Obstrutiva do Sono/genéticaRESUMO
OBJECTIVE: Medroxyprogesterone acetate (MPA) may increase the risk of atherosclerosis during hormone replacement therapy (HRT); therefore, the effect of progestogens other than MPA on atherosclerotic lesions requires evaluation. Adhesion of monocytes to vascular endothelial cells is an important early step in atherosclerosis progression. MCP-1 is a key chemokine that promotes monocyte migration and adhesion to vascular endothelial cells. In this study, we investigated the effects of dienogest (DNG), an alternative progestogen, on monocyte adhesion and cytokine expression in human umbilical vein endothelial cells (HUVECs). STUDY DESIGN: HUVECs were treated with DNG, natural progesterone, or MPA, followed by interleukin (IL)-1ß stimulation. The mRNA expression of adhesion molecules (E-selectin and ICAM-1) and cytokines (MCP-1 and IL-6) was examined using real-time PCR. A flow chamber system was used to examine the effect of DNG on the adhesion of U937 monocytic cells to monolayer HUVECs. RESULTS: Unlike MPA, DNG did not alter the mRNA expression of E-selectin, ICAM-1, MCP-1, and IL-6 in HUVECs. Moreover, it did not increase the number of monocytes adhering to HUVECs in the flow chamber system. However, MPA treatment significantly enhanced monocyte adhesion to HUVECs (p < 0.05). CONCLUSIONS: DNG had no effect on the mRNA expression of adhesion molecules and cytokines in HUVECs, as well as the monocyte adhesion to HUVECs, suggesting that DNG can be explored as an alternative to MPA for HRT.
Assuntos
Aterosclerose , Monócitos , Nandrolona/análogos & derivados , Humanos , Monócitos/metabolismo , Molécula 1 de Adesão Intercelular , Selectina E/genética , Selectina E/metabolismo , Interleucina-6/metabolismo , Molécula 1 de Adesão de Célula Vascular , Moléculas de Adesão Celular/metabolismo , Moléculas de Adesão Celular/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Citocinas/metabolismo , Aterosclerose/metabolismo , RNA Mensageiro/metabolismo , Expressão Gênica , Adesão Celular , Células CultivadasRESUMO
Inflammation is the body's biological reaction to endogenous and exogenous stimuli. Recent studies have demonstrated several anti-inflammatory properties of Ferula species. In this paper, we decided to study the anti-inflammatory effect of ethanolic extract of Ferula assafoetida oleo-gum-resin (asafoetida) against TNF-α-stimulated human umbilical vein endothelial cells (HUVECs). HUVECs were cultured in a flat-bottom plate and then treated with ethanolic extract of asafoetida (EEA, 0-500 µg/ml) and TNF-α (0-100 ng/ml) for 24 h. We used the MTT test to assess cell survival. In addition, the LC-MS analysis was performed to determine the active substances. HUVECs were pretreated with EEA and then induced by TNF-α. Intracellular reactive oxygen species (ROS) and adhesion of peripheral blood mononuclear cells (PBMCs) to HUVECs were evaluated with DCFH-DA and CFSE fluorescent probes, respectively. Gene expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin and surface expression of ICAM-1 protein were measured using real-time PCR and flow cytometry methods, respectively. While TNF-α significantly increased intracellular ROS formation and PBMC adhesion to TNF-α-induced HUVECs, the pretreatment of HUVECs with EEA (125 and 250 µg/ml) significantly reduced the parameters. In addition, EEA pretreatment decreased TNF-α-induced mRNA expression of VCAM-1 and surface protein expression of ICAM-1 in the target cells. Taken together, the results indicated that EEA prevented ROS generation, triggered by TNF-α, and inhibited the expression of VCAM-1 and ICAM-1, leading to reduced PBMC adhesion. These findings suggest that EEA can probably have anti-inflammatory properties.
Assuntos
Anti-Inflamatórios , Moléculas de Adesão Celular , Ferula , Células Endoteliais da Veia Umbilical Humana , Extratos Vegetais , Anti-Inflamatórios/farmacologia , Adesão Celular , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Selectina E/biossíntese , Selectina E/genética , Selectina E/imunologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Leucócitos Mononucleares/imunologia , Extratos Vegetais/farmacologia , Espécies Reativas de Oxigênio/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
Chronic vascular inflammation underlies many diseases, including atherosclerosis, autoimmune vasculitides and transplant rejection. The resolution of inflammation is critical for proper healing and restoration of homeostasis, but the timing and signaling mechanisms involved in the return to a non-inflamed state are not well understood. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells was measured in primary human aortic endothelium under chronic TNFα stimulation, and after short-term TNFα priming followed by withdrawal. The effects of NFκB, MAPK and JAK1/2 inhibitors on TNFα-induced gene expression were tested. The majority of inducible TNFα effectors, such as E-selectin, VCAM-1 and most chemokines, required continuous exposure for reinforcement of the altered phenotype, and were NFκB dependent. However, 3 h priming with TNFα induced late phase STAT activation and interferon response genes after 18 h, as well as enhanced ICAM-1, BST2 and CXCR3 ligand expression. Chronic activation was autonomous of continuous TNFα, and could be blocked by the JAK1/2 inhibitor ruxolitinib. The results demonstrate that NFκB is not a significant driver of the later phase of endothelial cell activation by TNFα, but that sustained inflammation is JAK1/2-dependent and characterized by adaptive chemokines.
Assuntos
Molécula 1 de Adesão Intercelular , Fator de Necrose Tumoral alfa , Selectina E/genética , Células Endoteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/genética , Interferons , Ligantes , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.
Assuntos
Selectina E , Neoplasias Pulmonares , Animais , Bortezomib/farmacologia , Antígeno CA-19-9/farmacologia , Adesão Celular , Selectina E/genética , Selectina E/metabolismo , Humanos , Ligantes , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Oligossacarídeos , Antígeno Sialil Lewis XRESUMO
Similar to the behavior of inflamed tubular epithelial cells, clear cell renal cell carcinoma (ccRCC) cells express death receptor 3 (DR3 or TNFSFR25) in situ, and expression increases with tumor grade. Surprisingly, E-selectin, which can be induced in endothelial cells by DR3 signaling, is also expressed by ccRCC cells and increases with tumor grade. In ccRCC organ cultures, addition of tumor necrosis factor-like 1A (TL1A or TNFSF15), the ligand for DR3, activates NF-κB and mitogen-activated protein kinases, induces both DR3 and E-selectin expression in an NF-κB-dependent manner, and promotes cell cycle entry. DR3 immunoprecipitated from ccRCC tissue contains sialyl Lewis X moieties (the ligand recognized by E-selectin), proximity ligation assays reveal DR3, and E-selectin interacts on ccRCC cells. Similar to that with the addition of TL1A, the addition of soluble E-selectin to ccRCC organ cultures activates NF-κB and mitogen-activated protein kinases in ccRCC cells and increases both DR3 and E-selectin expression and cell-cycle entry. In contrast, normal renal tubular epithelium, which poorly expresses DR3, is minimally responsive to either of these ligands. These data suggest a functional role for autocrine/paracrine DR3/E-selectin interactions in ccRCC and its progression, revealing a potential new target for therapeutic intervention.
Assuntos
Carcinoma de Células Renais , Selectina E , Neoplasias Renais , Membro 25 de Receptores de Fatores de Necrose Tumoral , Antígenos CD , Carcinoma de Células Renais/metabolismo , Selectina E/genética , Selectina E/metabolismo , Células Endoteliais/metabolismo , Feminino , Humanos , Neoplasias Renais/metabolismo , Ligantes , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Membro 25 de Receptores de Fatores de Necrose Tumoral/genética , Membro 25 de Receptores de Fatores de Necrose Tumoral/metabolismo , Membro 15 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismoRESUMO
Chronic low-grade retinal inflammation is an essential contributor to the pathogenesis of diabetic retinopathy (DR). It is characterized by increased retinal cell expression and secretion of a variety of inflammatory cytokines; among these, IL-1ß has the reputation of being a major driver of cytokine-induced inflammation. IL-1ß and other cytokines drive inflammatory changes that cause damage to retinal cells, leading to the hallmark vascular lesions of DR; these include increased leukocyte adherence, vascular permeability, and capillary cell death. Nuclear factor of activated T-cells (NFAT) is a transcriptional regulator of inflammatory cytokines and adhesion molecules and is expressed in retinal cells. Consequently, it may influence multiple pathogenic steps early in DR. We investigated the NFAT-dependency of IL-1ß-induced inflammation in human Müller cells (hMC) and human retinal microvascular endothelial cells (hRMEC). Our results show that an NFAT inhibitor, Inhibitor of NFAT-Calcineurin Association-6 (INCA-6), decreased IL-1ß-induced expression of IL-1ß and TNFα in hMC, while having no effect on VEGF, CCL2, or CCL5 expression. We also demonstrate that INCA-6 attenuated IL-1ß-induced increases of IL-1ß, TNFα, IL-6, CCL2, and CCL5 (inflammatory cytokines and chemokines), and ICAM-1 and E-selectin (leukocyte adhesion molecules) expression in hRMEC. INCA-6 similarly inhibited IL-1ß-induced increases in leukocyte adhesion in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Finally, INCA-6 rescued IL-1ß-induced permeability in both hRMEC monolayers in vitro and an acute model of retinal inflammation in vivo. Taken together, these data demonstrate the potential of NFAT inhibition to mitigate retinal inflammation secondary to diabetes.
Assuntos
Retinopatia Diabética/tratamento farmacológico , Inflamação/tratamento farmacológico , Interleucina-1beta/genética , Fatores de Transcrição NFATC/genética , Vasculite Retiniana/tratamento farmacológico , Inibidores de Calcineurina/farmacologia , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL5/genética , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Selectina E/genética , Células Endoteliais/efeitos dos fármacos , Células Ependimogliais/efeitos dos fármacos , Células Ependimogliais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/genética , Inflamação/patologia , Molécula 1 de Adesão Intercelular/genética , Interleucina-1beta/farmacologia , Fatores de Transcrição NFATC/antagonistas & inibidores , Retina/efeitos dos fármacos , Retina/patologia , Vasculite Retiniana/genética , Vasculite Retiniana/parasitologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia , Fator de Necrose Tumoral alfa/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Purpose: There are reports that a b-isoform of vascular endothelial growth factor-A 165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. Although these isoforms appear to have a different ability to activate the VEGF receptor 2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signaling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signaling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs). Methods: To determine full dose-response curves for the activation of MAPK (ERK1/2), AKT, and VEGFR2, direct in-cell western assays were developed using primary HRMECs. Potential differences in dose-response effects on gene expression markers related to endothelial cell and leukocyte adhesion (ICAM1, VCAM1, and SELE) and tight junctions (CLDN5 and OCLN) were tested with quantitative PCR. Results: Activation dose-response analysis revealed much stronger activation of MAPK, AKT, and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA 165 a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA 165 b. Similar maximum activation of MAPK was achieved by both isoforms at high concentrations. Maximum activation of AKT by VEGFA 165 b was only half of the maximum activation from VEGFA 165 a. At a lower intermediate dose, where VEGFA 165 a activated intracellular signaling stronger than VEGFA 165 b, the changes in VEGFA target gene expression were generally greater with VEGFA 165 a. Conclusions: In primary HRMECs, VEGFA 165 a could maximally activate MAPK and AKT at lower concentrations where VEGFA 165 b had relatively little effect. The timing for maximum activation of MAPK was similar for the isoforms, which is different from that reported for non-retinal endothelial cells. Although differences in VEGFA 165 a and VEGFA 165 b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ability to activate at least two key intracellular signaling pathways and VEGF-target gene expression in primary human retinal endothelial cells.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Vasos Retinianos/citologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Claudina-5/genética , Selectina E/genética , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Immunoblotting , Molécula 1 de Adesão Intercelular/genética , Ocludina/genética , Reação em Cadeia da Polimerase , Isoformas de Proteínas , Ativação Transcricional/fisiologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
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 eï¬ectively 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/metabolismoRESUMO
CD147 is involved in various physiological processes and plays important roles for tumor metastasis. Glycosylation of the protein determines numerous functions of CD147. Up to now, hardly any sensor has been developed for detecting glycosylation of CD147 in live cells. There is a pressing requirement of development of a selective and continuous biosensor for cell imaging. The emergence of gene-encoded fluorescence resonance energy transfer (FRET) sensor provides a new way to develop the sensors to analysts. We designed and constructed novel gene-encoded FRET proteins sensing glycosylation of CD147 by measuring FRET ratio of two intermolecular motifs. With the decrease of CD147 glycosylation level in cells, the FRET ratio increased significantly. The specificity of the sensor targeting to CD147 was also determined by siRNA interference experiment. Finally, continuous living cell image of deglycosylation process of CD147 using the newly developed sensor has been performed successfully. The work not only provides useful tools for analyzing glycosylation of CD147 in living cells, but also implicates alternative strategy for detecting other glycosylated proteins.
Assuntos
Proteínas de Bactérias/genética , Basigina/metabolismo , Técnicas Biossensoriais/métodos , Calgranulina B/genética , Selectina E/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/genética , Glicosilação , Células HeLa , HumanosRESUMO
BACKGROUND: Chronic idiopathic urticaria (CIU) represents a common skin disorder often characterized by mast cell activation and secretion of histamine and other proinflammatory factors. E-selectin (SELE) has been implicated in the pathogenesis of common inflammatory cutaneous disorders, while the role of SELE in CIU is yet to be fully understood. Thus, we aimed to investigate the mechanism by which SELE influences CIU in connection with the involvement of mast cells. METHODS: SELE expression was measured in blood samples obtained from CIU patients and normal individuals. A CIU mouse model was subsequently established by intradermally injecting a normal saline solution with ovalbumin IgE antiserum into the mice. Loss- and gain-of-function investigations were conducted on the mouse models. The number of degranulated mast cells and the amount of histamine release in vitro were determined. The levels of SELE, tumor necrosis factor (TNF)-α, homologous restriction factor (HRF), and interleukin (IL)-6 levels were determined. RESULTS: The CIU clinical samples exhibited upregulated SELE, while the CIU mice showed increased mast cell degranulation and an increased rate of histamine directional release, as well as an elevated expression of SELE, TNF-α, HRF, and IL-6. SELE silencing was found to decrease the number of degranulated mast cells and reduce the rate of histamine directional release, along with suppressed TNF-α, HRF, and IL-6 expression, in the serum of CIU mice. Ketotifen was observed to rescue the increased expression of TNF-α, HRF, and IL-6 caused by SELE overexpression. CONCLUSIONS: This study highlights the potential of SELE downregulation to repress inflammatory factor secretion caused by the accumulation of mast cells, which ultimately inhibits the development of CIU.
Assuntos
Urticária Crônica/etiologia , Urticária Crônica/metabolismo , Selectina E/genética , Regulação da Expressão Gênica , Mastócitos/imunologia , Mastócitos/metabolismo , Adolescente , Adulto , Animais , Biomarcadores , Urticária Crônica/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Selectina E/metabolismo , Feminino , Inativação Gênica , Humanos , Imuno-Histoquímica , Imunomodulação , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Adulto JovemRESUMO
Colorectal cancer is a common malign disease of the gastrointestinal tract. The cancer survival rate depends on the stage of the disease at detection time. It is well known that several molecular mechanisms are involved in cancer and some molecules might affect or modulate cancerogenesis. The aim of the study was to assess the levels of sICAM-1, sELAM-1, TNFα and sTNFR1 protein in tumor and corresponding normal mucosa in a group of patients with colorectal adenocarcinoma and also associations of these parameters with demographic and clinical profiles of the patients. Tissue specimens were obtained during resection of neoplastic lesions. Protein levels were assayed in tissue homogenates by ELISA. The protein level of sICAM-1 in tumor was significantly increased in comparison to the corresponding normal mucosa (80.06 ng/mg vs 69.53 ng/mg, p=0.02). Furthermore, a significant positive correlation between sICAM-1 and sTNFR1 proteins levels in tumor (rs=0.58, p<0.001) and in corresponding normal mucosa (rs=0.48, p<0.001) was found. Also, significant correlations in corresponding normal mucosa were found between sELAM-1 and sICAM-1 (rs=0.58, p<0.001) and between sTNFR1 and sELAM-1 (rs=0.57, p<0.001). Significantly higher level of sTNFR1 in corresponding normal mucosa samples of patients with distant metastases was observed (p=0.04). Obtained results suggest that sICAM-1 protein could be considered as colorectal cancer marker. Furthermore, sTNFR1 also has the potential to become a good prognostic marker used during monitoring of the patients. Nevertheless, a further study in this area to confirm this correlation is required.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Molécula 1 de Adesão Intercelular/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Selectina E/genética , Selectina E/metabolismo , Feminino , Expressão Gênica , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
OBJECTIVE: To assess the effects of resveratrol (RSV) on expression of adhesion molecules in endothelial progenitor cells (EPCs) following tumor necrosis factor-α (TNF-α) stimulation. METHODS: EPCs were treated with RSV and stimulated with TNF-α. A mononuclear cell (MNC) adhesion assay was used to assess the effects of RSV on TNF-α-induced MNC adhesion. Vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin expression levels and nuclear factor κB (NF-κB) activation were assessed by immunoblotting. RESULTS: MNC adhesion to TNF-α-treated EPCs and VCAM-1/ICAM-1/E-selectin levels in EPCs were increased following TNF-α stimulation and decreased following RSV treatment. TNF-α enhanced NF-κB inhibitor α (IκB-α) phosphorylation in the cytosol as well as nuclear NF-κB p65 levels, both of which were decreased by RSV. CONCLUSIONS: These findings provide new insights into RSV's anti-inflammatory and anti-atherosclerotic effects. RSV's mechanism of action might involve downregulation of VCAM-1, ICAM-1 and E-selectin by partial blockade of TNF-α-induced NF-κB activation and IκB-α phosphorylation in EPCs.
Assuntos
Células Progenitoras Endoteliais , Molécula 1 de Adesão de Célula Vascular , Células Cultivadas , Selectina E/genética , Células Progenitoras Endoteliais/metabolismo , Endotélio Vascular , Molécula 1 de Adesão Intercelular/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Resveratrol/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
γ-Glutamyl valine (γ-EV), commonly found in edible beans, was shown to reduce gastrointestinal inflammation via activation of calcium-sensing receptors (CaSRs). The present study aimed to evaluate the efficacy of γ-EV in modulating the tumor necrosis factor-α-induced inflammatory responses in endothelial cells (ECs) via CaSR-mediated pathways. Human aortic ECs (HAoECs) were pretreated (2 h) with γ-EV (0.01, 0.1, and 1 mM). 1 mM pretreatment of γ-EV significantly reduced the upregulation of inflammatory adhesion molecules, VCAM-1 and E-selectin, by 44.56 and 57.41%, respectively. The production of cytokines IL-8 and IL-6 was significantly reduced by 40 and 51%, respectively, with 1 mM pretreatment of γ-EV. Similarly, there was a significant reduction in chemokine MCP-1 from a positive control of 9.70 ± 0.52 to 6.6 ± 0.43 ng/mL, after γ-EV treatment. The anti-inflammatory effect of γ-EV was attenuated by the treatment of the CaSR-specific inhibitor, NPS-2143, suggesting the involvement of CaSR-mediated pathways. Further studies identified the critical role of key modulators, such as ß-arrestin2 and cyclic adenosine monophosphate response element-binding protein, in mediating the CaSR-dependent anti-inflammatory effect of γ-EV. Finally, the transport efficiency of γ-EV was evaluated through a monolayer of intestinal epithelial cells (Caco-2), and the apparent permeability (Papp) of the peptide was found to be 1.56 × 10-6 cm/s.
Assuntos
Células Endoteliais/efeitos dos fármacos , Peptídeos/farmacologia , Receptores de Detecção de Cálcio/imunologia , Fator de Necrose Tumoral alfa/imunologia , Células CACO-2 , Selectina E/genética , Selectina E/imunologia , Células Endoteliais/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Peptídeos/química , Receptores de Detecção de Cálcio/genética , Fator de Necrose Tumoral alfa/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
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 JovemRESUMO
OBJECTIVE: The direct actions of growth hormone (GH) in the development of atherosclerosis are unclear. The goal of this study was to characterize GH-induced changes in expression of signaling pathway elements and other proteins that may be related to atherosclerosis. METHODS: Human umbilical vein endothelial cells (HUVEC) and THP-1, a human acute monocytic leukemia cell line, were stimulated by exposure to 10-9 M or 10-8 M human GH with or without pretreatment with a mitogen-activated protein kinase kinase (MEK) 1 inhibitor. Levels of transcripts encoding vascular cell adhesion molecule (VCAM) -1, E-selectin, monocyte chemotactic protein (MCP-1), interleukin (IL) -6, and IL-8 were investigated by reverse transcription (RT) -PCR. For the quantitative adhesion assay, THP-1 cells or human primary monocytes were fluorescently labeled with 3'-O-acetyl-2',7'-bis(carboxyethyl) -4 diacetoxymethyl ester (BCECF/AM). HUVEC treated with human GH were co-incubated with BCECF-labeled THP-1 cells. One hour later, the number of BCECF-labeled THP-1 cells was assessed. An equivalent experiment was performed using BCECF-labeled primary monocytes, and the number of monocytes adhering to HUVEC was counted. RESULTS: Treatment with hGH increased the levels of E-selectin- and VCAM-1-encoding mRNAs in HUVEC. This effect was attenuated by pretreatment with a MEK1 inhibitor. Furthermore, hGH treatment increased adhesion of BCECF-labeled THP-1 cells or primary monocytes to HUVEC, and this effect was attenuated by pretreatment with a MEK1 inhibitor. CONCLUSIONS: VCAM-1 and E-selectin expression was stimulated by GH via the mitogen-activated protein kinase pathway, resulting in augmented adhesion of THP-1 cells and monocytes to HUVEC. These data suggested that GH directly stimulates the development of atherosclerosis.
Assuntos
Aterosclerose/patologia , Selectina E/metabolismo , Endotélio Vascular/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio do Crescimento Humano/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Aterosclerose/etiologia , Aterosclerose/metabolismo , Selectina E/genética , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Quinases Ativadas por Mitógeno/genética , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Monócitos/parasitologia , Monócitos/patologia , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Cell-cell interactions mediated by selectins and their ligand glycans play pivotal roles in a variety of biological processes represented by leukocyte recruitment to inflammatory sites, lymphocyte homing, and extravasation of cancer cells. The interactions are enhanced at least partly through the upregulation of the selectin-ligand glycan expression, which is observed, for instance, during the activation of leukocytes or epithelial-mesenchymal transition of cancer cells. Selectin-binding assays such as cell adhesion assay or rolling assay have long been used to directly evaluate the activity of these cells in the selectin-mediated processes. In this chapter, we introduce a highly quantitative assay by flow cytometry using recombinant selectin-Ig(Fc) chimera proteins, showing our procedure and tips for E-selectin-binding assay of colon cancer cells undergoing epithelial-mesenchymal transition.