Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.355
Filtrar
1.
Int J Mol Sci ; 22(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205118

RESUMO

During metastasis, cancer cells that originate from the primary tumor circulate in the bloodstream, extravasate, and form micrometastases at distant locations. Several lines of evidence suggest that specific interactions between cancer cells and endothelial cells, in particular tumor cell adhesion to the endothelium and transendothelial migration, play a crucial role in extravasation. Here we have studied the role of vascular endothelial (VE)-cadherin which is expressed aberrantly by breast cancer cells and might promote such interactions. By comparing different human breast cancer cell lines, we observed that the number of cancer cells that adhered to endothelium correlated with VE-cadherin expression levels. VE-cadherin silencing experiments confirmed that VE-cadherin enhances cancer cell adhesion to endothelial cells. However, in contrast, the number of cancer cells that incorporated into the endothelium was not dependent on VE-cadherin. Thus, it appears that cancer cell adhesion and incorporation are distinct processes that are governed by different molecular mechanisms. When cancer cells incorporated into the endothelial monolayer, they formed VE-cadherin positive contacts with endothelial cells. On the other hand, we also observed tumor cells that had displaced endothelial cells, reflecting either different modes of incorporation, or a temporal sequence where cancer cells first form contact with endothelial cells and then displace them to facilitate transmigration. Taken together, these results show that VE-cadherin promotes the adhesion of breast cancer cells to the endothelium and is involved in the initial phase of incorporation, but not their transmigration. Thus, VE-cadherin might be of relevance for therapeutic strategies aiming at preventing the metastatic spread of breast cancer cells.


Assuntos
Antígenos CD/genética , Neoplasias da Mama/genética , Caderinas/genética , Adesão Celular/genética , Endotélio Vascular/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Técnicas de Cocultura , Endotélio Vascular/patologia , Endotélio Vascular/ultraestrutura , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Imagem Molecular/métodos , Metástase Neoplásica
2.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067978

RESUMO

Galectin-3 (Gal-3) is a ß-galactoside-binding protein that influences various cell functions, including cell adhesion. We focused on the role of Gal-3 as an extracellular ligand mediating cell-matrix adhesion. We used human adipose tissue-derived stem cells and human umbilical vein endothelial cells that are promising for vascular tissue engineering. We found that these cells naturally contained Gal-3 on their surface and inside the cells. Moreover, they were able to associate with exogenous Gal-3 added to the culture medium. This association was reduced with a ß-galactoside LacdiNAc (GalNAcß1,4GlcNAc), a selective ligand of Gal-3, which binds to the carbohydrate recognition domain (CRD) in the Gal-3 molecule. This ligand was also able to detach Gal-3 newly associated with cells but not Gal-3 naturally present on cells. In addition, Gal-3 preadsorbed on plastic surfaces acted as an adhesion ligand for both cell types, and the cell adhesion was resistant to blocking with LacdiNAc. This result suggests that the adhesion was mediated by a binding site different from the CRD. The blocking of integrin adhesion receptors on cells with specific antibodies revealed that the cell adhesion to the preadsorbed Gal-3 was mediated, at least partially, by ß1 and αV integrins-namely α5ß1, αVß3, and αVß1 integrins.


Assuntos
Proteínas Sanguíneas/metabolismo , Adesão Celular , Junções Célula-Matriz/metabolismo , Galectinas/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Integrinas/metabolismo , Células-Tronco Mesenquimais/fisiologia , Sítios de Ligação , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Ligação Proteica
3.
Nat Commun ; 12(1): 3279, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078883

RESUMO

Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.


Assuntos
Anticorpos Neutralizantes/farmacologia , Cardiomiopatias/prevenção & controle , Cardiotoxicidade/prevenção & controle , Doxorrubicina/toxicidade , Raios gama/efeitos adversos , Molécula L1 de Adesão de Célula Nervosa/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/etiologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiotoxicidade/etiologia , Cardiotoxicidade/genética , Cardiotoxicidade/metabolismo , Estudos de Casos e Controles , Técnicas de Cocultura , Dano ao DNA , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/efeitos da radiação , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
4.
FASEB J ; 35(6): e21643, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33977574

RESUMO

The endothelial glycocalyx is a dynamic structure integral to blood vessel hemodynamics and capable of tightly regulating a range of biological processes (ie, innate immunity, inflammation, and coagulation) through dynamic changes in its composition of the brush structure. Evaluating the specific roles of the endothelial glycocalyx under a range of pathophysiologic conditions has been a challenge in vitro as it is difficult to generate functional glycocalyces using commonly employed 2D cell culture models. We present a new multi-height microfluidic platform that promotes the growth of functional glycocalyces by eliciting unique shear stress forces over a continuous human umbilical vein endothelial cell monolayer at magnitudes that recapitulate the physical environment in arterial, capillary and venous regions of the vasculature. Following 72 hours of shear stress, unique glycocalyx structures formed within each region that were distinct from that observed in short (3 days) and long-term (21 days) static cell culture. The model demonstrated glycocalyx-specific properties that match the characteristics of the endothelium in arteries, capillaries and veins, with respect to surface protein expression, platelet adhesion, lymphocyte binding and nanoparticle uptake. With artery-to-capillary-to-vein transition on a continuous endothelial monolayer, this in vitro platform is an improved system over static cell culture for more effectively studying the role of the glycocalyx in endothelial biology and disease.


Assuntos
Artérias/fisiologia , Capilares/fisiologia , Glicocálix/química , Glicocálix/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Estresse Mecânico , Veias/fisiologia , Hemodinâmica , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Microfluídica , Resistência ao Cisalhamento
5.
Nat Protoc ; 16(4): 2158-2189, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33790475

RESUMO

Owing to their high spatiotemporal precision and adaptability to different host cells, organ-on-a-chip systems are showing great promise in drug discovery, developmental biology studies and disease modeling. However, many current micro-engineered biomimetic systems are limited in technological application because of culture media mixing that does not allow direct incorporation of techniques from stem cell biology, such as organoids. Here, we describe a detailed alternative method to cultivate millimeter-scale functional vascularized tissues on a biofabricated platform, termed 'integrated vasculature for assessing dynamic events', that enables facile incorporation of organoid technology. Utilizing the 3D stamping technique with a synthetic polymeric elastomer, a scaffold termed 'AngioTube' is generated with a central microchannel that has the mechanical stability to support a perfusable vascular system and the self-assembly of various parenchymal tissues. We demonstrate an increase in user familiarity and content analysis by situating the scaffold on a footprint of a 96-well plate. Uniquely, the platform can be used for facile connection of two or more tissue compartments in series through a common vasculature. Built-in micropores enable the studies of cell invasion involved in both angiogenesis and metastasis. We describe how this protocol can be applied to create both vascularized cardiac and hepatic tissues, metastatic breast cancer tissue and personalized pancreatic cancer tissue through incorporation of patient-derived organoids. Platform assembly to populating the scaffold with cells of interest into perfusable functional vascularized tissue will require 12-14 d and an additional 4 d if pre-polymer and master molds are needed.


Assuntos
Vasos Sanguíneos/fisiologia , Dispositivos Lab-On-A-Chip , Organoides/fisiologia , Perfusão , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Tecidos Suporte/química
6.
J Biosci Bioeng ; 131(6): 686-695, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33775542

RESUMO

Angiogenesis is a pressing issue in tissue engineering associated with restoration of blood supply to ischemic tissues and promotion of rapid vascularization of tissue-engineered grafts. Fibroblast growth factor-2 (FGF-2) plays a vital role in processes such as angiogenesis and is an attractive candidate for tissue engineering. While skeletal muscle tissue engineering is established, the role of FGF-2 in endothelial function to promote angiogenesis after transplantation is unclear. Here, a culture system comprising a five-layered sheet of human skeletal muscle cells co-incubated on green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) mimicking in vivo angiogenesis was used to investigate the role of FGF-2 in vascularization of engineered tissues. The basal level of FGF-2 in cultured media of skeletal muscle cell sheets was undetectable. Therefore, cell sheets co-incubated with GFP-HUVECs were exogenously treated with 10 ng/mL FGF-2, and endothelial network formation was evaluated. After prolonged culture, the endothelial network length and connectivity increased following treatment with FGF-2 as compared with control treatment. The numbers of medium and long endothelial networks significantly increased inside the sheet longer than 0.2 and 0.4 cm, respectively, after FGF-2 treatment. Time-lapse microscopy monitoring dynamic endothelial behavior revealed that FGF-2-mediated maintenance of endothelial connection and retardation of endothelial network disconnection after 72 h. The present study suggests the precise role of FGF-2 in maintaining endothelial connection and the extent of the endothelial network in skeletal muscle cell sheets. This understanding can be applied to design in vitro pre-vascularized tissue and graft integration prospects.


Assuntos
Comunicação Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Meios de Cultura/química , Meios de Cultura/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Organoides/citologia , Organoides/efeitos dos fármacos , Organoides/fisiologia , Engenharia Tecidual/métodos
7.
Int J Mol Sci ; 22(5)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33671042

RESUMO

Short-chain fatty acids (e.g., butyrate and propionate) are able to diminish endothelial cell activation. The aim of this study was to investigate whether intracellular IL-33 mediates the effects of butyrate and propionate on TNFα-induced IL-8 production and vascular cell adhesion molecule-1 (VCAM-1) expression. In addition, it was investigated whether regulating NF-κB and MAPK signaling pathways are involved. Intracellular IL-33 was measured in human endothelial cells (HUVECs) pre-incubated for 24 h with butyrate (0.1 mM or 5 mM), propionate (0.3 mM or 10 mM), or trichostatin A (TSA, 0.5 µM) prior to TNFα (1 ng/mL) stimulation (24 h). The effects of butyrate, propionate, and TSA on TNFα-induced IL-8, vascular cell adhesion molecule-1 (VCAM-1), NF-κB, and MAPK signaling pathways in normal HUVECs and IL-33 siRNA (siIL-33)-transfected HUVECs were compared to study the role of IL-33 in the protective effects of butyrate and propionate. Endogenous IL-33 was highly expressed in the perinuclear in HUVECs, which was significantly reduced by TNFα stimulation. The TNFα-induced reduction in IL-33 was prevented by pre-incubation with butyrate or propionate. Butyrate (0.1 mM), propionate (0.3 mM), and TSA inhibited the IL-8 production and activation of NF-κB. Interestingly, this effect was not observed in siIL-33-transfected HUVECs. The effects of butyrate (5 mM), propionate (10 mM), and TSA (0.5 µM) on VCAM-1 expression and activation of MAPK signaling pathways were not affected by siIL-33 transfection. In conclusion, we showed that the inhibitory effects of butyrate and propionate on TNFα-induced IL-8 production were mediated by the HDACs/IL-33/NF-κB pathway, while their effects on VCAM-1 expression might be associated with the HDACs/MAPK signaling pathway, independently of IL-33.


Assuntos
Anti-Inflamatórios/farmacologia , Butiratos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Interleucina-33/metabolismo , Propionatos/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Histona Desacetilases/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Transdução de Sinais , Molécula 1 de Adesão de Célula Vascular/metabolismo
8.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673317

RESUMO

Vascular endothelial growth factor-A165 (VEGF-A165) and fibroblast growth factor-2 (FGF-2) are currently used for the functionalization of biomaterials designed for tissue engineering. We have developed a new simple method for heterologous expression and purification of VEGF-A165 and FGF-2 in the yeast expression system of Pichia pastoris. The biological activity of the growth factors was assessed in cultures of human and porcine adipose tissue-derived stem cells (ADSCs) and human umbilical vein endothelial cells (HUVECs). When added into the culture medium, VEGF-A165 stimulated proliferation only in HUVECs, while FGF-2 stimulated the proliferation of both cell types. A similar effect was achieved when the growth factors were pre-adsorbed to polystyrene wells. The effect of our recombinant growth factors was slightly lower than that of commercially available factors, which was attributed to the presence of some impurities. The stimulatory effect of the VEGF-A165 on cell adhesion was rather weak, especially in ADSCs. FGF-2 was a potent stimulator of the adhesion of ADSCs but had no to negative effect on the adhesion of HUVECs. In sum, FGF-2 and VEGF-A165 have diverse effects on the behavior of different cell types, which maybe utilized in tissue engineering.


Assuntos
Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células-Tronco/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Células-Tronco/citologia , Suínos , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/genética
9.
Int J Mol Sci ; 22(4)2021 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-33672986

RESUMO

Mesenchymal stromal cells derived from the fetal placenta, composed of an amnion membrane, chorion membrane, and umbilical cord, have emerged as promising sources for regenerative medicine. Here, we used next-generation sequencing technology to comprehensively compare amniotic stromal cells (ASCs) with chorionic stromal cells (CSCs) at the molecular and signaling levels. Principal component analysis showed a clear dichotomy of gene expression profiles between ASCs and CSCs. Unsupervised hierarchical clustering confirmed that the biological repeats of ASCs and CSCs were able to respectively group together. Supervised analysis identified differentially expressed genes, such as LMO3, HOXA11, and HOXA13, and differentially expressed isoforms, such as CXCL6 and HGF. Gene Ontology (GO) analysis showed that the GO terms of the extracellular matrix, angiogenesis, and cell adhesion were significantly enriched in CSCs. We further explored the factors associated with inflammation and angiogenesis using a multiplex assay. In comparison with ASCs, CSCs secreted higher levels of angiogenic factors, including angiogenin, VEGFA, HGF, and bFGF. The results of a tube formation assay proved that CSCs exhibited a strong angiogenic function. However, ASCs secreted two-fold more of an anti-inflammatory factor, TSG-6, than CSCs. In conclusion, our study demonstrated the differential gene expression patterns between ASCs and CSCs. CSCs have superior angiogenic potential, whereas ASCs exhibit increased anti-inflammatory properties.


Assuntos
Âmnio/citologia , Córion/citologia , Perfilação da Expressão Gênica/métodos , RNA-Seq/métodos , Células Estromais/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Feminino , Ontologia Genética , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Placenta/citologia , Placenta/metabolismo , Gravidez , Células THP-1
10.
Biomolecules ; 11(2)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562690

RESUMO

The endothelial to mesenchymal transition (End-MT) can be associated with vascular calcification, by providing mesengenic progenitors. In this study, we investigated a link between End-MT and the osteogenic process and explored the involvement of miR-30a-5p and miR-30d as potential regulators of these processes. End-MT was induced in Human Umbilical Vein Endothelial Cells (HUVEC) through transforming growth factor-ß1 (TGF-ß1), TGFß-3 and tumor necrosis factor-α (TNF-α), for 24 h and 6 days. End-MT mediators, mesenchymal and osteo/chondrogenic markers were analyzed through Real-Time PCR, immunofluorescence, flow cytometry and Western Blot. miR-30a-5p and miR-30d over-expression was carried out in HUVEC to explore their effects on End-MT and osteogenic differentiation. HUVEC at 24 h and 6 days gained mesenchymal morphology markers, including matrix metalloproteinase 9 (MMP-9), SLUG, VIMENTIN and α-smooth muscle actin (α-SMA), and a significant migratory potential, notably with TNF-α. After 6 days, the osteo/chondrogenic markers runt-related transcription factor 2 (RUNX-2) and SRY box transcription factor 9 (SOX-9) were upregulated. At this time point, miR-30a-5p and miR-30d decreased. Over-expression of miR-30a-5p and miR-30d affected End-MT mediators and the osteogenic potency in HUVEC, by reducing SLUG, VIMENTIN and RUNX-2. Our data suggest that End-MT represents a key link between inflammation and vascular calcification. Further, miR-30a-5p and miR-30d can regulate both the End-MT and the osteogenic processes, prompting future studies for exploring their potential use as therapeutic targets or biomarkers in vascular diseases.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta3/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Actinas/genética , Actinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Vimentina/genética , Vimentina/metabolismo
11.
Mol Med Rep ; 23(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33576443

RESUMO

Human umbilical vein endothelial cells (HUVECs) serve a critical role in maintaining normal vascular function. Lipopolysaccharide (LPS), which is released from pathogenic bacteria in the blood, induces HUVEC apoptosis and injury to cause vascular dysfunction and infectious vascular diseases. Procyanidin B2 (PB2) possesses numerous functions, including antioxidant, antitumor, anti­inflammatory and antiapoptosis effects, but the molecular mechanism is not completely understood. The present study investigated the effects of PB2 on LPS­induced cytotoxicity and apoptosis in HUVECs, as well as the underlying mechanisms. The effects of PB2 on LPS­mediated alterations to cytotoxicity, mitochondrial membrane potential, apoptosis were assessed by performing Cell Counting Kit­8, JC­1 fluorescence, Hoechst 33258 staining assays, respectively. IL­1ß, IL­6 and TNF­α mRNA expression and protein levels were measured by performing reverse transcription­quantitative PCR and ELISAs, respectively. Bcl­2, Bax, cleaved caspase­3, cleaved caspase­7, cleaved caspase­9, phosphorylated (p)­IκB­α, p­IκB­ß, p­NF­κB­p65 and total NF­κB p65 protein expression levels were determined via western blotting. NF­κB p65 nuclear translocation was assessed via immunofluorescence. PB2 pretreatment markedly attenuated LPS­induced cytotoxicity and apoptosis in HUVECs. PB2 also significantly downregulated the expression levels of IL­1ß, IL­6, TNF­α, Bax, cleaved caspase­3, cleaved caspase­7, cleaved caspase­9 and p­NF­κB­p65, but upregulated the expression levels of Bcl­2, p­IκB­α and p­IκB­ß in LPS­induced HUVECs. Moreover, PB2 markedly inhibited LPS­induced NF­κB p65 nuclear translocation in HUVECs. The results suggested that the potential molecular mechanism underlying PB2 was associated with the Bax/Bcl­2 and NF­κB signalling pathways. Therefore, PB2 may serve as a useful therapeutic for infectious vascular diseases.


Assuntos
Apoptose/efeitos dos fármacos , Biflavonoides/farmacologia , Catequina/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Proantocianidinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
12.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-33540846

RESUMO

The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.


Assuntos
Adesão Celular/fisiologia , Técnicas de Cultura de Células , Células Endoteliais da Veia Umbilical Humana/citologia , Apoptose , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Morte Celular , Divisão Celular , Meios de Cultivo Condicionados/química , Citocinas/análise , Epoprostenol/análise , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Mediadores da Inflamação/análise , Peptídeos e Proteínas de Sinalização Intercelular/análise , L-Lactato Desidrogenase/análise , Poliestirenos , Proteínas Recombinantes/farmacologia , Propriedades de Superfície , Tromboxano A2/análise , Fator de Necrose Tumoral alfa/farmacologia
13.
Proc Natl Acad Sci U S A ; 118(4)2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33468662

RESUMO

The two main blood flow patterns, namely, pulsatile shear (PS) prevalent in straight segments of arteries and oscillatory shear (OS) observed at branch points, are associated with atheroprotective (healthy) and atheroprone (unhealthy) vascular phenotypes, respectively. The effects of blood flow-induced shear stress on endothelial cells (ECs) and vascular health have generally been studied using human umbilical vein endothelial cells (HUVECs). While there are a few studies comparing the differential roles of PS and OS across different types of ECs at a single time point, there is a paucity of studies comparing the temporal responses between different EC types. In the current study, we measured OS and PS transcriptomic responses in human aortic endothelial cells (HAECs) over 24 h and compared these temporal responses of HAECs with our previous findings on HUVECs. The measurements were made at 1, 4, and 24 h in order to capture the responses at early, mid, and late time points after shearing. The results indicate that the responses of HAECs and HUVECs are qualitatively similar for endothelial function-relevant genes and several important pathways with a few exceptions, thus demonstrating that HUVECs can be used as a model to investigate the effects of shear on arterial ECs, with consideration of the differences. Our findings show that HAECs exhibit an earlier response or faster kinetics as compared to HUVECs. The comparative analysis of HAECs and HUVECs presented here offers insights into the mechanisms of common and disparate shear stress responses across these two major endothelial cell types.


Assuntos
Ciclo Celular/genética , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Redes e Vias Metabólicas/genética , Proteoma/genética , Estresse Mecânico , Fatores de Transcrição/genética , Aorta/citologia , Aorta/metabolismo , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Linhagem Celular , Proliferação de Células , Células Endoteliais/citologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Modelos Biológicos , Especificidade de Órgãos , Fenótipo , Proteoma/metabolismo , Transdução de Sinais , Biologia de Sistemas/métodos , Fatores de Transcrição/metabolismo
14.
Nat Commun ; 12(1): 257, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431893

RESUMO

Advances in deep learning technology have enabled complex task solutions. The accuracy of image classification tasks has improved owing to the establishment of convolutional neural networks (CNN). Cellular senescence is a hallmark of ageing and is important for the pathogenesis of ageing-related diseases. Furthermore, it is a potential therapeutic target. Specific molecular markers are used to identify senescent cells. Moreover senescent cells show unique morphology, which can be identified. We develop a successful morphology-based CNN system to identify senescent cells and a quantitative scoring system to evaluate the state of endothelial cells by senescence probability output from pre-trained CNN optimised for the classification of cellular senescence, Deep Learning-Based Senescence Scoring System by Morphology (Deep-SeSMo). Deep-SeSMo correctly evaluates the effects of well-known anti-senescent reagents. We screen for drugs that control cellular senescence using a kinase inhibitor library by Deep-SeSMo-based drug screening and identify four anti-senescent drugs. RNA sequence analysis reveals that these compounds commonly suppress senescent phenotypes through inhibition of the inflammatory response pathway. Thus, morphology-based CNN system can be a powerful tool for anti-senescent drug screening.


Assuntos
Forma Celular , Senescência Celular , Aprendizado Profundo , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Redes Neurais de Computação , beta-Galactosidase/metabolismo
15.
Carbohydr Polym ; 255: 117477, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33436240

RESUMO

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.


Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Heparitina Sulfato/biossíntese , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Complexo de Golgi/química , Complexo de Golgi/efeitos dos fármacos , Heparina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
16.
Chem Biol Interact ; 333: 109325, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33221320

RESUMO

Previous studies revealed that direct contact with graphene oxide (GO) induced cytotoxic effects, but the importance of involvement of metabolic pathways, in particular lipid metabolism pathways, might be overlooked. In this study, human umbilical vein endothelial cells (HUVECs) were exposed to GO with large size (denoted as GO-L) or small size (denoted as GO-S), and transcriptomics were used to understand the mechanisms of cytotoxicity of GO at systemic levels. It was shown that GO-L more significantly induced cytotoxicity compared with GO-S. Transcriptomic analysis revealed that compared with GO-S, GO-L had larger impact on gene ontology terms related with mitochondrial function as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related with cell death and growth. But GO-S showed greater influence on KEGG pathways related with lipid metabolism. Both types of GO showed minimal impact on oxidative stress but increased de novo lipogenesis protein fatty acid synthase (FASN). However, only GO-S significantly promoted acyl-CoA synthetase 3 (ACSL3), a key enzyme responsible for esterification of free fatty acids and lipid droplet biogenesis. Not surprisingly, GO-L but not GO-S impaired lipid droplet biogenesis, and increasing lipid levels by oleic acid or α-linolenic acid reduced the cytotoxicity of GO-L to HUVECs. Combined, the results from this study suggested that impaired lipid droplet biogenesis was involved in GO-induced cytotoxicity in HUVECs, and inducing lipid droplet biogenesis could prevent the cytotoxicity of GO.


Assuntos
Citotoxinas/toxicidade , Perfilação da Expressão Gênica , Grafite/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Coenzima A Ligases/genética , Ácido Graxo Sintase Tipo I/genética , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
17.
Lasers Med Sci ; 36(1): 83-90, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32304000

RESUMO

The aim of the present study was to investigate the influence of low-level red (660 nm) and infrared (780 nm) laser with four different radiance exposures on human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs (1.5 × 104) were incubated in 96-well culture plates. The cells were maintained in M199 medium supplemented with 20% fetal bovine serum, 1% antibiotic (penicillin), 1% anti-mycotic (Fungizone), and 1% endothelial cell growth supplement. After centrifugation, irradiations (660/780 nm, 40 mW, 1, 5, 10, and 20 J/cm2, 1 s, 5 s, 10 s, and 20 s, respectively, total energy 0.4 J, 2 J, 4 J, and 8 J, and beam spot size at target 0.04 cm2) were performed at the bottom of Falcon tubes such that the laser beam directly reached the cell without passing through the culture medium. The cells were divided into groups based on radiant exposures. Cell viability and protein concentration were verified after 1, 2, 3, 6, 8, and 10 days. Red laser increased the cell viability and protein concentration in all groups (three-way ANOVA, p < 0.05) beginning on the second day. The greatest peak compared with the control was found when the radiant exposure was 5 J/cm2 and 10 J/cm2. Infrared laser inhibited cell viability and modulated the protein concentration in the cells, with the highest peak protein concentration found on the second day in the group with radiant exposure of 1 J/cm2 and 10 J/cm2 (three-way ANOVA, p < 0.05). Red laser increased the viability and concentration of total proteins in HUVECs, whereas infrared laser had an inhibitory effect on cell viability, while maintaining the total protein concentration similar to that found in the control group.


Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Terapia com Luz de Baixa Intensidade , Ciclo Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Meios de Cultura/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Lasers
18.
Methods Mol Biol ; 2206: 39-46, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754809

RESUMO

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.


Assuntos
Bioensaio/métodos , Técnicas de Cocultura/métodos , Neovascularização Patológica/patologia , Comunicação Celular/fisiologia , Células Cultivadas , Fibroblastos/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos
19.
Methods Mol Biol ; 2206: 47-56, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754810

RESUMO

The development and maintenance of a functioning vascular system is a critical function for many aspects of tissue growth and regeneration. Vascular endothelial cell in vitro co-culture spheroids are self-organized cell composites that have the capacity to recapitulate the three-dimensional tissue microenvironment. These spheroid testing platforms aim to better understand the mechanisms of functional tissue and how new therapeutic agents can drive these 3D co-culture processes. Here we describe direct cell-cell 3D endothelial co-culture spheroid methods, to examine the physiological spatial growth and cell-cell interaction of vascular cells and surrounding native tissue cells in the formation of vascular networks within spheroids and the potential to regenerate tissue.


Assuntos
Técnicas de Cocultura/métodos , Células Endoteliais da Veia Umbilical Humana/citologia , Esferoides Celulares/citologia , Comunicação Celular/fisiologia , Células Cultivadas , Humanos , Engenharia Tecidual/métodos
20.
Methods Mol Biol ; 2206: 57-66, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32754811

RESUMO

The construction of vascular networks is essential for developing functional organ/tissue constructs in terms of oxygen and nutrient supply. Although recent advances in microfluidic techniques have allowed for the construction of microvascular networks using microfluidic devices, their structures cannot be maintained for extended periods of time due to a lack of perivascular cells. To construct long-lasting microvascular networks, it is important that perivascular cells are present to provide structural support to vessels, because in vivo microvessels are covered by perivascular cells and stabilized. Here, we describe a microfluidic cell culture platform for the construction of microvascular networks with supportive perivascular cells. Our results showed that microvascular networks covered by pericyte-like perivascular cells formed in a microfluidic device and their structures were maintained for at least 3 weeks in vitro.


Assuntos
Células Endoteliais da Veia Umbilical Humana/citologia , Células-Tronco Mesenquimais/citologia , Microfluídica/métodos , Células Cultivadas , Técnicas de Cocultura/métodos , Humanos , Microvasos/citologia , Pericitos/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...