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1.
J Transl Med ; 19(1): 383, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496870

RESUMO

BACKGROUND: Antiangiogenic therapy has increasingly become an important strategy for the treatment of colorectal cancer. Recent studies have shown that the tumour microenvironment (TME) promotes tumour angiogenesis. Bufalin is an active antitumour compound whose efficacy has been indicated by previous studies. However, there are very few studies on the antiangiogenic effects of bufalin. METHODS: Herein, human umbilical vein endothelial cell (HUVEC) tube formation, migration and adhesion tests were used to assess angiogenesis in vitro. Western blotting and quantitative PCR were used to detect relevant protein levels and mRNA expression levels. A subcutaneous xenograft tumour model and a hepatic metastasis model were established in mice to investigate the influence of bufalin on angiogenesis mediated by the TME in vivo. RESULTS: We found that angiogenesis mediated by cells in the TME was significantly inhibited in the presence of bufalin. The results demonstrated that the proangiogenic genes in HUVECs, such as VEGF, PDGFA, E-selectin and P-selectin, were downregulated by bufalin and that this downregulation was mediated by inhibition of the STAT3 pathway. Overexpression of STAT3 reversed the inhibitory effects of bufalin on angiogenesis. Furthermore, there was little reduction in angiogenesis when bufalin directly acted on the cells in the tumour microenvironment. CONCLUSION: Our findings demonstrate that bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway in vascular endothelial cells, revealing that bufalin may be used as a new antiangiogenic adjuvant therapy medicine to treat colorectal cancer.


Assuntos
Neoplasias Hepáticas , Microambiente Tumoral , Inibidores da Angiogênese/farmacologia , Animais , Bufanolídeos , Movimento Celular , Proliferação de Células , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Camundongos , Camundongos Nus , Neovascularização Patológica/tratamento farmacológico , Fator de Transcrição STAT3/metabolismo
2.
Int J Mol Sci ; 22(16)2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34445234

RESUMO

Abiotrophia defectiva is a nutritionally variant streptococci that is found in the oral cavity, and it is an etiologic agent of infective endocarditis. We have previously reported the binding activity of A. defectiva to fibronectin and to human umbilical vein endothelial cells (HUVECs). However, the contribution of some adhesion factors on the binding properties has not been well delineated. In this study, we identified DnaK, a chaperon protein, as being one of the binding molecules of A. defectiva to fibronectin. Recombinant DnaK (rDnaK) bound immobilized fibronectin in a concentration-dependent manner, and anti-DnaK antiserum reduced the binding activity of A. defectiva with both fibronectin and HUVECs. Furthermore, DnaK were observed on the cell surfaces via immune-electroscopic analysis with anti-DnaK antiserum. Expression of IL-8, CCL2, ICAM-1, and VCAM-1 was upregulated with the A. defectiva rDnaK treatment in HUVECs. Furthermore, TNF-α secretion of THP-1 macrophages was also upregulated with the rDnaK. We observed these upregulations in rDnaK treated with polymyxin B, but not in the heat-treated rDnaK. The findings show that A. defectiva DnaK functions not only as an adhesin to HUVECs via the binding to fibronectin but also as a proinflammatory agent in the pathogenicity to cause infective endocarditis.


Assuntos
Abiotrophia/metabolismo , Aderência Bacteriana , Proteínas de Bactérias/metabolismo , Fibronectinas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Abiotrophia/genética , Proteínas de Bactérias/genética , Proteínas de Choque Térmico HSP70/genética , Células Endoteliais da Veia Umbilical Humana/microbiologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Inflamação/microbiologia
3.
Int J Mol Sci ; 22(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34445773

RESUMO

Inadequate vessel maintenance or growth causes ischemia in diseases such as myocardial infarction, stroke, and neurodegenerative disorders. Therefore, developing an effective strategy to salvage ischemic tissues using a novel compound is urgent. Drug repurposing has become a widely used method that can make drug discovery more efficient and less expensive. Additionally, computational virtual screening tools make drug discovery faster and more accurate. This study found a novel drug candidate for pro-angiogenesis by in silico virtual screening. Using Gene Expression Omnibus (GEO) microarray datasets related to angiogenesis studies, differentially expressed genes were identified and characteristic direction signatures extracted from GEO2EnrichR were used as input data on L1000CDS2 to screen pro-angiogenic molecules. After a thorough review of the candidates, a list of compounds structurally similar to TWS-119 was generated using ChemMine Tools and its clustering toolbox. ChemMine Tools and ChemminR structural similarity search tools for small-molecule analysis and clustering were used for second screening. A molecular docking simulation was conducted using AutoDock v.4 to evaluate the physicochemical effect of secondary-screened chemicals. A cell viability or toxicity test was performed to determine the proper dose of the final candidate, ellipticine. As a result, we found ellipticine, which has pro-angiogenic effects, using virtual computational methods. The noncytotoxic concentration of ellipticine was 156.25 nM. The phosphorylation of glycogen synthase kinase-3ß was decreased, whereas the ß-catenin expression was increased in human endothelial cells treated with ellipticine. We concluded that ellipticine at sublethal dosage could be successfully repositioned as a pro-angiogenic substance by in silico virtual screening.


Assuntos
Elipticinas/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neovascularização Patológica/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Descoberta de Drogas/métodos , Reposicionamento de Medicamentos/métodos , Expressão Gênica/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Neovascularização Patológica/metabolismo , Ligação Proteica/efeitos dos fármacos , beta Catenina/metabolismo
4.
Molecules ; 26(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34443423

RESUMO

Chronic liver inflammation has become a major global health concern. In the absence of clinical surrogate markers to diagnose inflammatory liver disease, the intervention with effective drugs in modern medicine tends to be late. In Sri Lanka, traditional medical practitioners prescribe herbal preparations from Osbeckia octandra for the prevention and treatment of liver disorders. To test the efficacy of such treatments, we have administered thioacetamide (TAA) to male Wistar rats to induce chronic liver damage (disease control; DC) and examined how various leaf extracts: crude leaf suspension (CLS), boiled leaf extract (BLE), sonicated leaf extract (SLE), methanol leaf extract (MLE) and hexane leaf extract (HLE) of O. octandra ameliorate TAA-induced liver disease. The CLS, BLE and SLE treatments in cirrhotic rats significantly attenuated disease-related changes, such as liver weight and hepato-enzymes. The mRNA levels of Tnf-α were significantly decreased by 3.6, 10 and 3.9 times in CLS, BLE and SLE compared to DC. The same treatments resulted in significantly lower (19.5, 4.2 and 2.4 times) α-Sma levels compared to DC. In addition, Tgf-ß1 and Vegf-R2 mRNA expressions were significantly lower with the treatments. Moreover, BLE expressed a strong anti-angiogenic effect. We conclude that CLS, BLE and SLE from O. octandra have potent hepatic anti-fibrotic effects in TAA-induced liver cirrhosis.


Assuntos
Cirrose Hepática Experimental/tratamento farmacológico , Melastomataceae/química , Neovascularização Patológica/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Folhas de Planta/química , Citocinas/genética , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática Experimental/sangue , Neovascularização Patológica/sangue , Tamanho do Órgão/efeitos dos fármacos , Extratos Vegetais/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Tioacetamida , Regulação para Cima/efeitos dos fármacos , Água , Perda de Peso/efeitos dos fármacos
5.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445337

RESUMO

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFß2 and IL1ß. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.


Assuntos
Transdiferenciação Celular/genética , Rim/patologia , MicroRNAs/fisiologia , Miocárdio/patologia , Animais , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Fibrose/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Rim/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia
6.
J Med Food ; 24(8): 852-859, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34382871

RESUMO

CYJ-27, a synthetic analog of decursin, prevents the generation of proinflammatory cytokines and oxidative stress. In this study, the effects of CYJ-27 on the regulation of inducible nitric oxide synthase (iNOS), heme oxygenase (HO)-1, and cyclooxygenase (COX-)2 were characterized in lipopolysaccharide (LPS)-treated human umbilical vein endothelial cells (HUVECs). In addition, the effects of CYJ-27 on the production of iNOS and representative proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, were tested in the lung tissues of LPS-treated mice. CYJ-27 promoted the expression of HO-1, suppressed NF-κB-luciferase activity, and reduced COX-2/PGE2 and iNOS/NO, resulting in a diminution in phosphorylated-STAT-1. Furthermore, CYJ-27 promoted the nuclear translocation of Nrf2, enhanced the combination of Nrf2 to antioxidant response elements, and diminished IL-1ß production in LPS-activated HUVECs. CYJ-27-downregulated iNOS/NO expression was rescued after the RNAi suppression of HO-1. In LPS-treated mice, CYJ-27 significantly diminished iNOS production in the lung tissues and TNF-α expression in the bronchoalveolar lavage fluid. These findings indicate that CYJ-27 exerts anti-inflammatory activities by regulating iNOS through downregulation of both NF-κB activation and phosphorylated-STAT-1. Hence, it can act as a template for the development of novel substances to treat inflammatory diseases.


Assuntos
Inflamação , NF-kappa B , Animais , Benzopiranos , Butiratos , Ciclo-Oxigenase 2/metabolismo , Regulação para Baixo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/genética , Lipopolissacarídeos , Camundongos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo
7.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360944

RESUMO

Endothelial and epithelial barrier function is crucial for the maintenance of physiological processes. The barrier paracellular permeability depends on the composition and spatial distribution of the cell-to-cell tight junctions (TJ). Here, we provide an experimental workflow that yields several layers of physiological data in the setting of a single endothelial cell monolayer. Human umbilical vein endothelial cells were grown on Transwell filters. Transendothelial electrical resistance (TER) and 10 kDa FITC dextran flux were measured using Alanyl-Glutamine (AlaGln) as a paracellular barrier modulator. Single monolayers were immunolabelled for Zonula Occludens-1 (ZO-1) and Claudin-5 (CLDN5) and used for automated immunofluorescence imaging. Finally, the same monolayers were used for single molecule localization microscopy (SMLM) of ZO-1 and CLDN5 at the nanoscale for spatial clustering analysis. The TER increased and the paracellular dextran flux decreased after the application of AlaGln and these functional changes of the monolayer were mediated by an increase in the ZO-1 and CLDN5 abundance in the cell-cell interface. At the nanoscale level, the functional and protein abundance data were accompanied by non-random increased clustering of CLDN5. Our experimental workflow provides multiple data from a single monolayer and has wide applicability in the setting of paracellular studies in endothelia and epithelia.


Assuntos
Permeabilidade Capilar , Junções Íntimas/metabolismo , Claudina-5/metabolismo , Dextranos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteína da Zônula de Oclusão-1/metabolismo
8.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360960

RESUMO

BACKGROUND/AIMS: Estrogen has been reported to have beneficial effects on vascular biology through direct actions on endothelium. Together with transcription factors, miRNAs are the major drivers of gene expression and signaling networks. The objective of this study was to identify a comprehensive regulatory network (miRNA-transcription factor-downstream genes) that controls the transcriptomic changes observed in endothelial cells exposed to estradiol. METHODS: miRNA/mRNA interactions were assembled using our previous microarray data of human umbilical vein endothelial cells (HUVEC) treated with 17ß-estradiol (E2) (1 nmol/L, 24 h). miRNA-mRNA pairings and their associated canonical pathways were determined using Ingenuity Pathway Analysis software. Transcription factors were identified among the miRNA-regulated genes. Transcription factor downstream target genes were predicted by consensus transcription factor binding sites in the promoter region of E2-regulated genes by using JASPAR and TRANSFAC tools in Enrichr software. RESULTS: miRNA-target pairings were filtered by using differentially expressed miRNAs and mRNAs characterized by a regulatory relationship according to miRNA target prediction databases. The analysis identified 588 miRNA-target interactions between 102 miRNAs and 588 targets. Specifically, 63 upregulated miRNAs interacted with 295 downregulated targets, while 39 downregulated miRNAs were paired with 293 upregulated mRNA targets. Functional characterization of miRNA/mRNA association analysis highlighted hypoxia signaling, integrin, ephrin receptor signaling and regulation of actin-based motility by Rho among the canonical pathways regulated by E2 in HUVEC. Transcription factors and downstream genes analysis revealed eight networks, including those mediated by JUN and REPIN1, which are associated with cadherin binding and cell adhesion molecule binding pathways. CONCLUSION: This study identifies regulatory networks obtained by integrative microarray analysis and provides additional insights into the way estradiol could regulate endothelial function in human endothelial cells.


Assuntos
Estradiol/farmacologia , Estrogênios/farmacologia , Redes Reguladoras de Genes , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Transcriptoma , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360750

RESUMO

Tissue regeneration depends on the complex processes of angiogenesis, inflammation and wound healing. Regarding muscle tissue, glucocorticoids (GCs) inhibit pro-inflammatory signalling and angiogenesis and lead to muscle atrophy. Our hypothesis is that the synthetic GC dexamethasone (dex) impairs angiogenesis leading to muscle atrophy or inhibited muscle regeneration. Therefore, this study aims to elucidate the effect of dexamethasone on HUVECs under different conditions in mono- and co-culture with myoblasts to evaluate growth behavior and dex impact with regard to muscle atrophy and muscle regeneration. Viability assays, qPCR, immunofluorescence as well as ELISAs were performed on HUVECs, and human primary myoblasts seeded under different culture conditions. Our results show that dex had a higher impact on the tube formation when HUVECs were maintained with VEGF. Gene expression was not influenced by dex and was independent of cells growing in a 2D or 3D matrix. In co-culture CD31 expression was suppressed after incubation with dex and gene expression analysis revealed that dex enhanced expression of myogenic transcription factors, but repressed angiogenic factors. Moreover, dex inhibited the VEGF mediated pro angiogenic effect of myoblasts and inhibited expression of angiogenic inducers in the co-culture model. This is the first study describing a co-culture of human primary myoblast and HUVECs maintained under different conditions. Our results indicate that dex affects angiogenesis via inhibition of VEGF release at least in myoblasts, which could be responsible not only for the development of muscle atrophy after dex administration, but also for inhibition of muscle regeneration after vascular damage.


Assuntos
Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Mioblastos Esqueléticos/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Mioblastos Esqueléticos/citologia
10.
Stem Cell Res Ther ; 12(1): 415, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34294121

RESUMO

BACKGROUND: Osteogenesis is tightly coupled with angiogenesis during bone repair and regeneration. However, the underlying mechanisms linking these processes remain largely undefined. The present study aimed to test the hypothesis that epidermal growth factor-like domain-containing protein 6 (EGFL6), an angiogenic factor, also functions in bone marrow mesenchymal stem cells (BMSCs), playing a key role in the interaction between osteogenesis and angiogenesis. METHODS: We evaluated how EGFL6 affects angiogenic activity of human umbilical cord vein endothelial cells (HUVECs) via proliferation, transwell migration, wound healing, and tube-formation assays. Alkaline phosphatase (ALP) and Alizarin Red S (AR-S) were used to assay the osteogenic potential of BMSCs. qRT-PCR, western blotting, and immunocytochemistry were used to evaluate angio- and osteo-specific markers and pathway-related genes and proteins. In order to determine how EGFL6 affects angiogenesis and osteogenesis in vivo, EGFL6 was injected into fracture gaps in a rat tibia distraction osteogenesis (DO) model. Radiography, histology, and histomorphometry were used to quantitatively evaluate angiogenesis and osteogenesis. RESULTS: EGFL6 stimulated both angiogenesis and osteogenic differentiation through Wnt/ß-catenin signaling in vitro. Administration of EGFL6 in the rat DO model promoted CD31hiEMCNhi type H-positive capillary formation associated with enhanced bone formation. Type H vessels were the referred subtype involved during DO stimulated by EGFL6. CONCLUSION: EGFL6 enhanced the osteogenic differentiation potential of BMSCs and accelerated bone regeneration by stimulating angiogenesis. Thus, increasing EGFL6 secretion appeared to underpin the therapeutic benefit by promoting angiogenesis-coupled bone formation. These results imply that boosting local concentrations of EGFL6 may represent a new strategy for the treatment of compromised fracture healing and bone defect restoration.


Assuntos
Osteogênese por Distração , Osteogênese , Animais , Diferenciação Celular , Células Cultivadas , Consolidação da Fratura , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Ratos , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
11.
Nat Commun ; 12(1): 4556, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315880

RESUMO

The potential applications of covalent organic frameworks (COFs) can be further developed by encapsulating functional nanoparticles within the frameworks. However, the synthesis of monodispersed core@shell structured COF nanocomposites without agglomeration remains a significant challenge. Herein, we present a versatile dual-ligand assistant strategy for interfacial growth of COFs on the functional nanoparticles with abundant physicochemical properties. Regardless of the composition, geometry or surface properties of the core, the obtained core@shell structured nanocomposites with controllable shell-thickness are very uniform without agglomeration. The derived bowl-shape, yolk@shell, core@satellites@shell nanostructures can also be fabricated delicately. As a promising type of photosensitizer for photodynamic therapy (PDT), the porphyrin-based COFs were grown onto upconversion nanoparticles (UCNPs). With the assistance of the near-infrared (NIR) to visible optical property of UCNPs core and the intrinsic porosity of COF shell, the core@shell nanocomposites can be applied as a nanoplatform for NIR-activated PDT with deep tissue penetration and chemotherapeutic drug delivery.


Assuntos
Estruturas Metalorgânicas/química , Nanopartículas/química , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Feminino , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Ligantes , Camundongos Endogâmicos BALB C , Nanocompostos/química , Nanopartículas/ultraestrutura , Fotoquimioterapia , Porfirinas/química , Dióxido de Silício/química , Tela Subcutânea/efeitos dos fármacos , Tela Subcutânea/patologia
12.
Chem Biol Interact ; 345: 109572, 2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34217687

RESUMO

Oxidized low-density lipoprotein (ox-LDL) not only causes hyperlipidemia and contributes to atherosclerosis but also induces the endothelial dysfunction that leads to cardiovascular diseases. The nuclear factor-kappa B (NF-κB) pathway plays a key role in many chronic disorders and is a transcriptional factor in various inflammatory responses. The present study aimed to investigate the synergistic effects of pelargonic acid vanillylamide (PAVA) and rosuvastatin (RSV) on ox-LDL-induced inflammatory responses in human vascular endothelial cells (HUV-EC-C). HUV-EC-C were pretreated with PAVA or RSV and their combination for 2 h followed by ox-LDL for 24 h. The MTT assay was used to measure mitochondrial function. The DCFH-DA assay was used to evaluate oxidative phosphorylation, and western blotting was used to measured NF-κB/NLRP3 and related signaling pathways in HUV-EC-C. Ox-LDL induced lectin-type oxidized LDL receptor 1 (LOX-1) expression, NADPH oxidase 4 activation, and the overexpression of reactive oxygen species, which were inhibited by pretreatment with the combination of PAVA and RSV. Moreover, PAVA and RSV inhibited ox-LDL-induced NF-κBp65 activation. Ox-LDL induced NF-κB/NLRP3 pathway activation by inducing C-reactive protein expression, NLRP3 activation, caspase-1 activation, and IL-1ß secretion, which were inhibited by pretreatment with the combination of PAVA and RSV. The combination of PAVA and RSV reduced ox-LDL-induced recruitment of monocytes to the site of inflammation, inhibited activation of the NLRP3 inflammasome, and ameliorated the impairment of cell-cell junctions through the NF-κB pathway. Our results suggest that the synergistic effects of PAVA and RSV provide a novel mechanism for the treatment of cardiovascular diseases.


Assuntos
Benzilaminas/farmacologia , Citoproteção/efeitos dos fármacos , Ácidos Graxos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Junções Intercelulares/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Caspase 1/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamassomos/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
13.
Signal Transduct Target Ther ; 6(1): 266, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253708

RESUMO

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1ß and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.


Assuntos
COVID-19 , Células Endoteliais da Veia Umbilical Humana , Fatores de Transcrição Kruppel-Like/biossíntese , SARS-CoV-2 , COVID-19/genética , COVID-19/metabolismo , COVID-19/patologia , COVID-19/prevenção & controle , Citocinas/biossíntese , Citocinas/genética , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/virologia , Humanos , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Fatores de Transcrição Kruppel-Like/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese , Molécula 1 de Adesão de Célula Vascular/genética
14.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206648

RESUMO

The aim of this study was to investigate the C-terminal cleavage of (pyr)-apelin-13 in human endothelial cells with respect to the role and subcellular location of prolyl carboxypeptidase (PRCP). Human umbilical vein and aortic endothelial cells, pre-treated with prolyl carboxypeptidase-inhibitor compound 8o and/or angiotensin converting enzyme 2 (ACE2)-inhibitor DX600, were incubated with (pyr)-apelin-13 for different time periods. Cleavage products of (pyr)-apelin-13 in the supernatant were identified by mass spectrometry. The subcellular location of PRCP was examined via immunocytochemistry. In addition, PRCP activity was measured in supernatants and cell lysates of LPS-, TNFα-, and IL-1ß-stimulated cells. PRCP cleaved (pyr)-apelin-13 in human umbilical vein and aortic endothelial cells, while ACE2 only contributed to this cleavage in aortic endothelial cells. PRCP was found in endothelial cell lysosomes. Pro-inflammatory stimulation induced the secretion of PRCP in the extracellular environment of endothelial cells, while its intracellular level remained intact. In conclusion, PRCP, observed in endothelial lysosomes, is responsible for the C-terminal cleavage of (pyr)-apelin-13 in human umbilical vein endothelial cells, while in aortic endothelial cells ACE2 also contributes to this cleavage. These results pave the way to further elucidate the relevance of the C-terminal Phe of (pyr)-apelin-13.


Assuntos
Aorta/citologia , Carboxipeptidases/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Linhagem Celular , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mediadores da Inflamação/metabolismo , Peptídeos/sangue , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
15.
Int J Nanomedicine ; 16: 4631-4642, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34262273

RESUMO

Purpose: Antibiotic-resistant bacteria are pathogens that have emerged as a serious public health risk. Thus, there is an urgent need to develop a new generation of anti-bacterial materials to kill antibiotic-resistant bacteria. Methods: Nanosilver-decorated mesoporous organosilica nanoparticles (Ag-MONs) were fabricated for co-delivery of gentamicin (GEN) and nanosilver. After investigating the glutathione (GSH)-responsive matrix degradation and controlled release of both GEN and silver ions, the anti-bacterial activities of Ag-MONs@GEN were systematically determined against several antibiotic-susceptible and antibiotic-resistant bacteria including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. Furthermore, the cytotoxic profiles of Ag-MONs@GEN were evaluated. Results: The GEN-loaded nanoplatform (Ag-MONs@GEN) showed glutathione-responsive matrix degradation, resulting in the simultaneous controlled release of GEN and silver ions. Ag-MONs@GEN exhibited excellent anti-bacterial activities than Ag-MONs and GEN alone via inducing ROS generation, especially enhancing synergetic effects against four antibiotic-resistant bacteria including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis. Moreover, the IC50 values of Ag-MONs@GEN in L929 and HUVECs cells were 313.6 ± 15.9 and 295.7 ± 12.3 µg/mL, respectively, which were much higher than their corresponding minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. Conclusion: Our study advanced the development of Ag-MONs@GEN for the synergistic and safe treatment of antibiotic-resistant bacteria.


Assuntos
Resistência Microbiana a Medicamentos/efeitos dos fármacos , Gentamicinas/farmacologia , Glutationa/química , Nanopartículas/química , Dióxido de Silício/química , Prata/química , Animais , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Liberação Controlada de Fármacos , Sinergismo Farmacológico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Nanopartículas/ultraestrutura
16.
Front Immunol ; 12: 623610, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093519

RESUMO

Severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) strongly hampered the broad clinical applicability of chimeric antigen receptor T cell (CAR-T) therapy. Vascular endothelial activation has been suggested to contribute to the development of CRS and ICANS after CAR-T therapy. However, therapeutic strategies targeting endothelial dysfunction during CAR-T therapy have not been well studied yet. Here, we found that tumor necrosis factor α (TNFα) produced by CAR-T cells upon tumor recognition and interleukin 1ß (IL1ß) secreted by activated myeloid cells were the main cytokines in inducing endothelial activation. Therefore, we investigated the potential effectiveness of TNFα and IL1ß signaling blockade on endothelial activation in CAR-T therapy. The blockade of TNFα and IL1ß with adalimumab and anti-IL1ß antibody respectively, as well as the application of focal adhesion kinase (FAK) inhibitor, effectively ameliorated endothelial activation induced by CAR-T, tumor cells, and myeloid cells. Moreover, adalimumab and anti-IL1ß antibody exerted synergistic effect on the prevention of endothelial activation induced by CAR-T, tumor cells, and myeloid cells. Our results indicate that TNFα and IL1ß blockade might have therapeutic potential for the treatment of CAR-T therapy-associated CRS and neurotoxicity.


Assuntos
Adalimumab/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Imunoterapia Adotiva , Interleucina-1beta/antagonistas & inibidores , Células Mieloides/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/efeitos dos fármacos , Inibidores do Fator de Necrose Tumoral/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Antígenos CD19/genética , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/metabolismo , Síndrome da Liberação de Citocina/prevenção & controle , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Imunoterapia Adotiva/efeitos adversos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Células Mieloides/imunologia , Células Mieloides/metabolismo , Síndromes Neurotóxicas/imunologia , Síndromes Neurotóxicas/metabolismo , Síndromes Neurotóxicas/prevenção & controle , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
17.
Biomed Pharmacother ; 140: 111788, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34062414

RESUMO

Complex regional pain syndrome (CRPS) is a chronic pain condition characterized by inflammation and debilitating pain. CRPS patients with pain refractory to more conventional analgesics can be treated with subanesthetic doses of ketamine. Our previous studies found that poor responders to ketamine had a 22-fold downregulation of the miRNA hsa-miR-605 in blood prior to ketamine treatment. Hence, we sought to investigate the functional significance of miR-605 downregulation and its impact on target gene expression, as investigating target mRNAs of differentially expressed miRNAs can provide important insights on aberrant gene expression that may contribute to disease etiology. Using a bioinformatics prediction, we identified that miR-605 can target the proinflammatory chemokine CXCL5, which plays a role in leukocyte recruitment and activation. We hypothesized that downregulation of miR-605 in poor responders to ketamine could increase CXCL5 expression and thereby contribute to inflammation in these patients. We confirmed that miR-605 regulates CXCL5 by using a miRNA mimic and inhibitor in human primary endothelial cells. Inhibition of miR-605 increased CXCL5 secretion and migration of human monocytic cells, thereby demonstrating a functional impact of miR-605 on chemotaxis. Additionally, CXCL5 mRNA was upregulated in whole blood from poor responders to ketamine, and CXCL5 protein was increased in plasma from CRPS patients. Thus, our studies suggest that miR-605 regulation of CXCL5 can regulate inflammation.


Assuntos
Quimiocina CXCL5/imunologia , Síndromes da Dor Regional Complexa/imunologia , MicroRNAs/imunologia , Analgésicos/uso terapêutico , Movimento Celular , Quimiocina CXCL5/sangue , Quimiocina CXCL5/genética , Síndromes da Dor Regional Complexa/sangue , Síndromes da Dor Regional Complexa/tratamento farmacológico , Síndromes da Dor Regional Complexa/genética , Regulação para Baixo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Ketamina/uso terapêutico , MicroRNAs/metabolismo , Monócitos/imunologia , Monócitos/fisiologia , Células THP-1 , Fator de Necrose Tumoral alfa/farmacologia
19.
Genes Cells ; 26(8): 611-626, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34081835

RESUMO

Serum/glucocorticoid-regulated kinase 1 (SGK1) is predominantly expressed in endothelial cells of mouse embryos, and Sgk1 null mice show embryonic lethality due to impaired vascular formation. However, how the SGK1 expression is controlled in developing vasculature remains unknown. In this study, we first identified a proximal endothelial enhancer through lacZ reporter mouse analyses. The mouse Sgk1 proximal enhancer was narrowed down to the 5' region of the major transcription initiation site, while a human corresponding region possessed relatively weak activity. We then searched for distal enhancer candidates using in silico analyses of publicly available databases for DNase accessibility, RNA polymerase association and chromatin modification. A region approximately 500 kb distant from the human SGK1 gene was conserved in the mouse, and the mouse and human genomic fragments drove transcription restricted to embryonic endothelial cells. Minimal fragments of both proximal and distal enhancers had consensus binding elements for the ETS transcription factors, which were essential for the responsiveness to ERG, FLI1 and ETS1 proteins in luciferase assays and the endothelial lacZ reporter expression in mouse embryos. These results suggest that endothelial SGK1 expression in embryonic vasculature is maintained through at least two ETS-regulated enhancers located in the proximal and distal regions.


Assuntos
Endotélio Vascular/metabolismo , Elementos Facilitadores Genéticos , Proteínas Imediatamente Precoces/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Cromatina/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/embriologia , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Proteínas Imediatamente Precoces/genética , Camundongos , Proteínas Oncogênicas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteína Proto-Oncogênica c-ets-1/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , Sítio de Iniciação de Transcrição , Regulador Transcricional ERG/metabolismo
20.
Int J Nanomedicine ; 16: 4045-4061, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34163158

RESUMO

Purpose: Previous studies demonstrated the possibility of targeting tumor-angiogenic endothelial cells with positively charged nanocarriers, such as cationic liposomes. We investigated the active targeting potential of positively charged nanoparticles in combination with the heat-induced drug release function of thermosensitive liposomes (TSL). This novel dual-targeted approach via cationic TSL (CTSL) was thoroughly explored using either a novel synthetic phospholipid 1,2-dipalmitoyl-sn-glycero-3-phosphodiglycerol (DPPG2) or a conventional polyethylene glycol (PEG) surface modification. Anionic particles containing either DPPG2 or PEG were also included in the study to highlight difference in tumor enrichment driven by surface charge. With this study, we aim to provide a deep insight into the main differences between DPPG2- and PEG-functionalized liposomes, focusing on the delivery of a well-known cytotoxic drug (doxorubicin; DOX) in combination with local hyperthermia (HT, 41-43°C). Materials and Methods: DPPG2- and PEG-based cationic TSLs (PG2-CTSL/PEG-CTSL) were thoroughly analyzed for size, surface charge, and heat-triggered DOX release. Cancer cell targeting and DOX delivery was evaluated by FACS, fluorescence imaging, and HPLC. In vivo particle behavior was analyzed by assessing DOX biodistribution with local HT application in tumor-bearing animals. Results: The absence of PEG in PG2-CTSL promoted more efficient liposome-cell interactions, resulting in a higher DOX delivery and cancer cell toxicity compared with PEG-CTSL. By exploiting the dual-targeting function of CTSLs, we were able to selectively trigger DOX release in the intracellular compartment by HT. When tested in vivo, local HT promoted an increase in intratumoral DOX levels for all (C)TSLs tested, with DOX enrichment factors ranging from 3 to 14-fold depending on the type of formulation. Conclusion: Cationic particles showed lower hemocompatibility than their anionic counterparts, which was partially mitigated when PEG was grafted on the liposome surface. DPPG2-based anionic TSL showed optimal local drug delivery compared to all other formulations tested, demonstrating the potential advantages of using DPPG2 lipid in designing liposomes for tumor-targeted applications.


Assuntos
Comunicação Celular , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Fosfatidilgliceróis/química , Polietilenoglicóis/química , Temperatura , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Doxorrubicina/farmacocinética , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Concentração Inibidora 50 , Espaço Intracelular/metabolismo , Lipossomos , Polietilenoglicóis/administração & dosagem , Ratos , Propriedades de Superfície , Distribuição Tecidual/efeitos dos fármacos
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