RESUMO
PURPOSE: Little is known about the molecular interactions among inflammatory responses that damage venous endothelial cells (vECs) during venous-to-arterial flow transition in vein graft diseases. Because arterial flow triggers excessive autophagy and inflammation in vECs, this study aimed to investigate the mediator of inflammation and methods to prevent vEC damage. METHODS: Arterial laminar shear stress (ALSS; 12 dynes/cm2) was applied to vECs via in vitro and ex vivo perfusion systems. Inflammation in vECs was measured using inflammatory protein markers, NFκB translocation, cyclooxygenase-2 (COX-2) and COX-2 and NFκB promoter assays. The involvement of microRNA-4488 (miR-4488) was measured and confirmed by altering the specific miR using a miR-4488 mimic or inhibitor. The potential anti-inflammatory drugs and/or nitric oxide (NO) donor L-arginine (L-Arg) to prevent damage to vECs under ALSS was investigated. RESULTS: ALSS triggered reactive oxygen species production, excessive autophagy, COX-2 protein expression, and NFκB translocation during vEC inflammation. Reduction in miR-4488 expression was detected in inflamed vECs treated with LPS, lipopolysaccharide (LPS) TNFα, and ALSS. Transfection of miR-4488 mimic (50 nM) prior to ALSS application inhibited the accumulation of inflammatory proteins as well as the translocation of NFκB. Combined treatment of vECs with COX-2-specific inhibitor (SC-236) and L-Arg alleviated the ALSS-induced inflammatory responses. Protective effects of the combined treatment on vECs against ALSS-induced damage were abolished by the application of miR-4488 inhibitor. CONCLUSION: We showed that ALSS triggered the COX-2/NFκB pathway to induce vEC inflammation with a reduction in miR-4488. Combination of SC-236 and L-Arg prevented ALSS-induced vEC damage, thus, shows high potential for preventing vein graft diseases.
Assuntos
Endotélio Vascular/metabolismo , Mediadores da Inflamação/metabolismo , MicroRNAs/biossíntese , NF-kappa B/metabolismo , Anti-Inflamatórios/farmacologia , Autofagia/efeitos dos fármacos , Ponte de Artéria Coronária/efeitos adversos , Vasos Coronários/fisiopatologia , Ciclo-Oxigenase 2/efeitos dos fármacos , Hemodinâmica , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipopolissacarídeos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Veia Safena/fisiopatologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/biossínteseRESUMO
BACKGROUND: This study is to investigate the role and mechanism of mir-5189-3p in deep vein thrombosis (DVT) in lower extremity. METHODS: The blood samples were collected from Kazakh patients with DVT in lower extremity and were subjected to microRNA sequencing. Bioinformatics were used to identify mir-5189-3p and its target genes. Dual luciferase reporter assay was used to determine the regulatory effect of mir-5189-3p on JAG1. SD rats were randomly divided into normal control, DVT model, hsa-miR-5189-3p mimics and hsa-miR-5189-3p negative control groups. HE staining was used to observe the pathological changes. TUNEL method was used to observe apoptosis. Western blot was used to detect Bax and Bcl-2 protein expression. Real-time quantitative PCR was used to detect JAG1, Notch1 and Hes1 mRNA. RESULTS: The target of Has-miR-5189-3p was JAG1. Co-transfection of miR-5189-3p mimics and pmirGLO/JAG1 wild-type plasmid induced significantly decreased luciferase activity. In hsa-miR-5189-3p mimics and hsa-miR-5189-3p negative control groups, there were more nucleated cells in the thrombus tissues, and the organization degree obviously increased. Signs of blood flow recanalization were observed. The apoptosis of hsa-miR-5189-3p mimics and hsa-miR-5189-3p negative control groups was lower than that in DVT model group. Furthermore, mir-5189-3p mimics significantly increased the mRNA levels of JAG1, Notch1 and Hes1. Additionally, mir-5189-3p mimics significantly increased Bcl-2 while decreased Bax protein. CONCLUSIONS: mir-5189-3p could inhibit apoptosis and promote thrombus organization in DVT possibly via Notch signaling pathway. Mir-5189-3p can be used as a potential target for DVT treatment.
Assuntos
MicroRNAs/metabolismo , Veia Cava Inferior/metabolismo , Trombose Venosa/metabolismo , Animais , Apoptose , Modelos Animais de Doenças , Células HEK293 , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , MicroRNAs/sangue , MicroRNAs/genética , MicroRNAs/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos Sprague-Dawley , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo , Veia Cava Inferior/patologia , Trombose Venosa/sangue , Trombose Venosa/genética , Trombose Venosa/patologia , Proteína X Associada a bcl-2/metabolismoRESUMO
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/farmacologiaRESUMO
The development processes of arteries and veins are fundamentally different, leading to distinct differences in anatomy, structure, and function as well as molecular profiles. Understanding the complex interaction between genetic and epigenetic pathways, as well as extracellular and biomechanical signals that orchestrate arterial venous differentiation, is not only critical for the understanding of vascular diseases of arteries and veins but also valuable for vascular tissue engineering strategies. Recent research has suggested that certain transcriptional factors not only control arterial venous differentiation during development but also play a critical role in adult vessel function and disease processes. This review summarizes the signaling pathways and critical transcription factors that are important for arterial versus venous specification. We focus on those signals that have a direct relation to the structure and function of arteries and veins, and have implications for vascular disease processes and tissue engineering applications.
Assuntos
Artérias/patologia , Endotélio Vascular/patologia , Engenharia Tecidual/métodos , Veias/patologia , Animais , Fenômenos Biomecânicos , Diferenciação Celular , Células Endoteliais/citologia , Epigênese Genética , Humanos , Hipóxia , Camundongos , Células-Tronco Pluripotentes/citologia , Transdução de Sinais , Células-Tronco/citologia , Fatores de Transcrição , Peixe-ZebraRESUMO
WHAT IS KNOWN AND OBJECTIVE: Essential hypertension (EH) is a common disease exhibiting large individual difference in occurrence, development and treatment response. Genetic factors are implicated in the development and progression of EH. This study aimed to explore the association between NPR3 single nucleotide polymorphism rs2270915 (A/G, Asn521Asp) and the risk of EH in a Chinese Han population by a case-control study. METHODS: The study was a single-centre, case-control trial, in which a total of 287 EH patients and 289 age- and sex-matched healthy controls were enrolled. The inclusion criteria were as follows: Han Chinese origin, male or female patients, systolic blood pressure (SBP) ≥140 mm Hg and/or diastolic blood pressure (DBP) ≥90 mm Hg. The healthy controls were subjects without histories of cardiovascular or cerebrovascular diseases. NPR3 rs2270915 polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). In addition, primary human umbilical vein endothelial cells (HUVECs) were isolated from 19 fresh human umbilical cords and cultured. Atrial natriuretic peptide (ANP) concentration in cell medium was determined by enzyme-linked immunosorbent assay (ELISA). NPR3 mRNA expression was determined by real-time semi-quantitative PCR. RESULTS AND DISCUSSION: No significant difference in genotype distribution of NPR3 rs2270915 polymorphism was observed between cases and controls (P>.05). Patients carrying the rs2270915 G allele showed decreased SBP, and the difference was marginal. As compared with cells carrying the rs2270915 AA genotype, those with the AG genotype showed significantly lower NPR3 mRNA expression levels (P<.05) and lower medium ANP concentration (P<.001). WHAT IS NEW AND CONCLUSION: This study suggested that NPR3 rs2270915 polymorphism was associated with decreased SBP level marginally in EH patients in a Chinese Han population, and the polymorphism may function through decreasing NPR3 mRNA expression and ANP level.
Assuntos
Fator Natriurético Atrial/metabolismo , Pressão Sanguínea/genética , Hipertensão Essencial/genética , Receptores do Fator Natriurético Atrial/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Povo Asiático/genética , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Predisposição Genética para Doença , Genótipo , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection. METHODS: The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs. RESULTS: A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01). CONCLUSION: DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Assuntos
Autofagia , Dengue , Células Endoteliais da Veia Umbilical Humana , Proteoma , Humanos , Morte Celular , Núcleo Celular , Células Endoteliais da Veia Umbilical Humana/virologiaRESUMO
Cyclophosphamide (CPA) is one of the most successful anticancer prodrugs that becomes effective after biotransformation in the liver resulting in the toxic metabolite acrolein. Cancer is often accompanied by thromboembolic events, which might be a result of dysfunctional endothelial cells due to CPA treatment. Here, the effect of 1â¯mM CPA or acrolein (10/50/100/500⯵M) on human umbilical vein endothelial cells (HUVECs) was analyzed after two days of treatment. The addition of CPA or 10⯵M acrolein did not affect HUVECs. However, concentrations of 100⯵M and 500⯵M acrolein significantly reduced the number of adherent cells by 86⯱â¯13% and 99⯱â¯1% and cell viability by 51⯱â¯29% and 93⯱â¯8% compared to the control. Moreover, pronounced stress fibers as well as multiple nuclei were observed and von Willebrand factor (vWF) was completely released. Lactate dehydrogenase was 8.5⯱â¯7.0-fold and 252.9⯱â¯42.9-fold increased showing a loss of cell membrane integrity. The prostacyclin and thromboxane secretion was significantly increased by the addition of 500⯵M acrolein (43.1⯱â¯17.6-fold and 246.4⯱â¯106.3-fold) indicating cell activation/pertubation. High doses of acrolein led to HUVEC death and loss of vWF production. This effect might be associated with the increased incidence of thromboembolic events in cancer patients treated with high doses of CPA.
Assuntos
Acroleína/toxicidade , Antineoplásicos Alquilantes/toxicidade , Ciclofosfamida/toxicidade , Células Endoteliais/efeitos dos fármacos , Pró-Fármacos/toxicidade , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Epoprostenol/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Cultura Primária de Células , Tromboxanos/metabolismo , Fator de von Willebrand/metabolismoRESUMO
OBJECTIVE: This study aimed to investigate the effects of neuromuscular blocking drugs on the viability of human umbilical vein endothelial cells (HUVECs) and to investigate whether they cause vascular complications due to cell proliferation. METHODS: HUVECs were cultivated with 5% CO2 at 37°C in a predefined supplemented medium over 7 days until confluence of cell monolayers. Assays were conducted during the exponential growth phase. Suxamethonium chloride, vecuronium bromide, atracurium besylate, and rocuronium bromide were used at concentrations of 10-5, 10-6, and 10-7 M in proliferation assays in which cells were incubated with these drugs for 24, 48, and 72 hours. All experiments were performed in four replicates. RESULTS: The neuromuscular blocking drugs used had comparable effects on the survivability of HUVECs. Overall, no significant difference was observed in the survivability of HUVECs in a dose-dependent manner after exposure to the study drugs. However, some significant differences in the viability of HUVECs were found among the different measurement times. CONCLUSIONS: The findings of the current study support the safety of the studied neuromuscular blocking drugs in clinically relevant concentrations regarding their effects on endothelial cell proliferation.
Assuntos
Bloqueio Neuromuscular , Preparações Farmacêuticas , Androstanóis , Atracúrio , Células Endoteliais da Veia Umbilical Humana , Humanos , Brometo de VecurônioRESUMO
OBJECTIVE@#To analyze the differentially phosphorylated proteins in DENV-2-infected human umbilical venous endothelial cells (HUVECs) and explore the possible pathogenic mechanism of DENV-2 infection.@*METHODS@#The total proteins were extracted from DENV-2-infected HUVECs and blank control HUVEC using SDT lysis method. The phosphorylated proteins were qualitatively and quantitatively analyzed using tandem mass spectrometry (TMT). The identified differentially phosphorylated proteins were analyzed by bioinformatics analyses such as subcellular localization analysis, GO enrichment analysis, KEGG pathway analysis and protein-protein interaction (PPI) analysis. Western blotting was used to detect the expressions of phosphorylated Jun, map2k2 and AKT1 proteins in DENV-2-infected HUVECs.@*RESULTS@#A total of 2918 modified peptides on 1385 different proteins were detected, and among them 1346 were significantly upregulated (FC > 1.2, P < 0.05) and 1572 were significantly downregulated (FC < 0.83, P < 0.05). A total of 49 phosphorylated conserved motifs were obtained by amino acid conservative motif analysis. The most abundant differentially phosphorylated peptides in protein domain analysis included RNA recognition motif, protein kinase domain and PH domain. Subcellular localization analysis showed that the differentially modified peptides were mainly localized in the nucleus and cytoplasm. GO enrichment and KEGG pathway analysis showed that the differential peptides were mainly enriched in the regulation of stimulation response, biosynthesis of small molecules containing nuclear bases, and migration of phagosomes and leukocytes across the endothelium. PPI and KEGG joint analysis showed that the up-regulated and down-regulated differentially phosphorylated proteins were enriched in 15 pathways. In DENV-2-infected HUVECs, Western blotting detected differential expressions of phosphorylated proteins related with the autophagy pathway, namely JUN, MAP2K2 and AKT1, and among them p-JUN was significantly down-regulated and p-AKT1 and p-MAP2K2 were significantly upregulated (P < 0.01).@*CONCLUSION@#DENV-2 infected HUVECs show numerous differentially expressed proteins. The downregulation of p-JUN and upregulation of p-MAP2K2 and p-AKT1 suggest their potential roles in regulating autophagy, which is probably involved in the mechanism of DENV-2 infection.
Assuntos
Humanos , Autofagia , Morte Celular , Núcleo Celular , Células Endoteliais da Veia Umbilical Humana/virologia , Dengue , ProteomaRESUMO
OBJECTIVE: Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation. METHODS: By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer. Cultured HUVEC were analysed for adhesion molecule expression by means of flow cytometry, while the morphological changes were evaluated by means of SEM. Cell adhesion was evaluated by means of flow cytometry and both OM and SEM. RESULTS: HUVEC expressed under resting conditions the adhesion molecules ICAM-1, VCAM-1 and E-selectin and their expression was upregulated by stimulation with TNF-α (0.1-10ng/ml) as well as with LPS (1µg/ml). SEM analysis showed that stimulation with both stimuli profoundly affect cell morphology. Flow cytometric evaluation of cell adhesion showed that the ability of cells to adhere to HUVEC monolayer was quite different in the two preparations, with the lowest adhesion for FB in all the cell subsets analysed. Finally, isolated granulocytes were able to adhere to HUVEC monolayer more than cells identified in FB or BC and the adhesion was increased during activation of HUVEC with 10ng/ml of TNF-α. CONCLUSION: Our data showed that cell manipulation necessary for the isolation of specific immune cells from whole blood profoundly affect the ability of these cells to adhere to the HUVEC monolayer although their functional properties remain unchanged.
Assuntos
Moléculas de Adesão Celular/metabolismo , Adesão Celular , Separação Celular/métodos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neutrófilos/metabolismo , Antígenos CD18/metabolismo , Adesão Celular/efeitos dos fármacos , Forma Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração , Técnicas de Cocultura , Selectina E/metabolismo , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/ultraestrutura , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Lipopolissacarídeos/farmacologia , Microscopia Eletrônica de Varredura , Neutrófilos/ultraestrutura , Fenótipo , Fator de Necrose Tumoral alfa/farmacologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case-control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09E-5≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78E-5≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36E-4≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52E-6≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL of early-onset PE compared with spontaneous preterm birth. Implications of the tissue-specific variations in epigenetic programming for circadian performance and long-term health need further investigation.
Assuntos
Relógios Circadianos/genética , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Ritmo Circadiano/genética , Metilação de DNA , Epigênese Genética , Placenta/metabolismo , Pré-Eclâmpsia/genética , Adulto , Idade de Início , Estudos de Casos e Controles , Células Cultivadas , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/sangue , Ilhas de CpG , Feminino , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Recém-Nascido , Países Baixos , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/diagnóstico , Gravidez , Adulto JovemRESUMO
Objective@#To explore the inhibitory effect of exosomes secreted by human umbilical cord mesenchymal stem cells(HUCMSC) on apoptosis of human umbilical vein endothelial cells(HUVEC) after model group(oxygen-glucose deprivation reoxygenation), and to clarify its possible mechanism.@*Methods@#Human umbilical cord mesenchymal stem cells were cultured. The collected cell supernatant was stored in a centrifugal tube. The exosomes secreted by human umbilical cord mesenchymal stem cells were extracted by ultracentrifugation and identified. Human umbilical vein endothelial cells were randomly divided into control group, model group and different concentrations of HUCMSC-EXO(20 μg/ml, 40 μg/ml, 60 μg/ml) treatment groups(adding HUCMSC-EXO into the model group) . The morphological changes of HUVEC cells in each group were observed by inverted phase contrast microscope, and the proliferation inhibition rate of HUVEC in each group was measured by CCK-8 reagent. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3, Bax, Bcl-2 and hypoxia-associated protein hypoxia inducible factor 1α(HIF-1α). Inhibitor(HIF-1α inhibitor) + model group and HUCMSC-EXO + inhibitor + model group were added on the basis of the above experiments. Western blot analysis was performed to observe the effects of HUCMSC-EXO, inhibitor and both of them on HIF-1α and Bax expressions in HUVEC.@*Results@#HUCMSC-EXO was successfully extracted and identified. Compared with the control group, the volume of HUVEC in the model group and the HUCMSC-EXO group with different concentrations decreased, became round, connected and evacuated, and the growth state was poor under the inverted phase contrast microscope.CCK-8 detection showed that the cell viability in the HUCMSC-EXO group was significantly higher than that in the model group, the difference was statistically significant (t=9.23, P<0.05). Western blot analysis showed that compared with the control group, the expression levels of Caspase-3 ((0.296±0.038), (0.879±0.088); t=14.92, P<0.05), Bax((0.234±0.034), (0.762±0.084); t=14.36, P<0.05) of HUVEC in the model group were up-regulated, and the expression level of Bcl-2 was down-regulated ((0.863±0.103), (0.387±0.059); t=9.85, P<0.05), with statistically significant differences. Compared with the model group, the expression levels of Caspase-3( (0.586±0.075); t=6.24, P<0.05), Bax((0.311±0.055); t=11.01, P<0.05) and Bcl-2((0.665±0.071); t=7.45, P<0.05) of HUVEC in the HUCMSC-EXO treatment group were down-regulated and the differences were statistically significant. Inhibitor intervention experiments showed that there were no significant differences between the inhibitor+ model group and HUCMSC-EXO+ inhibitor+ model group in the expression of HIF-1α protein ((0.348±0.055), (0.388±0.077); t=1.04, P>0.05)and Bax protein ((0.363±0.069), (0.370±0.064); t=0.18, P>0.05). But both of them were down-regulated compared with the model group (HIF-1α protein (0.919±0.064), Bax protein (0.902±0.071)), the differences were significant( t=13.56, t=13.03, both P<0.05).@*Conclusion@#HUCMSC-EXO has a protective effect on OGD/R model of HUVEC, and its mechanism may be related to the down-regulation of HIF-1α expression.
RESUMO
AIM:To investigate the effects of dehydroepiandrosterone(DHEA)on the expression of intercellu-lar adhesion molecule-1(ICAM-1)induced by high lipid levels in rabbit aorta and human umbilical venous endothelial cells(HUVECs),and the effects of all-trans retinoic acid(ATRA)in this process.METHODS: For in vitro experi-ments,the cultured HUVEC were divided into control group,oxidized low-density lipoprotein(ox-LDL)group,ox-LDL+DHEA group,ox-LDL+DHEA+ATRA group and DHEA group.The HUVECs in all groups were treated with the corre-sponding reagents for 24 h.The expression of ICAM-1 at mRNA and protein levels in all groups were determined by RT-PCR and ELISA,respectively.For in vivo experiments,the rabbits were divided into control group,high lipid group,high lipid+DHEA group,high lipid+DHEA+ATRA group and DHEA group.The rabbits in all groups were fed with the cor-responding diets for 10 weeks.The expression of ICAM-1 in the rabbit aorta at mRNA and protein levels was determined by RT-PCR and immunohistochemistry.RESULTS:The expression of ICAM-1 in the HUVECs in ox-LDL group was signifi-cantly increased compared with control group(P<0.05).Compared with ox-LDL group,the expression of ICAM-1 in ox-LDL+DHEA group was obviously decreased(P<0.05).The expression of ICAM-1 was similar in both control group and DHEA group(P>0.05).The expression of ICAM-1 was similar in both ox-LDL+DHEA group and ox-LDL+DHEA+ATRA group(P>0.05).The expression of ICAM-1 in the rabbit aorta in high lipid group was significantly increased com-pared with control group(P<0.05).Compared with high lipid group, the expression of ICAM-1 in high lipid+DHEA group was obviously decreased(P<0.05).No remarkable difference in the expression of ICAM-1 between control group and DHEA group was observed(P>0.05), so did between high lipid +DHEA group and high lipid +DHEA+ATRA group(P>0.05).CONCLUSION:DHEA inhibits high lipid-induced ICAM-1 expression in rabbit aorta and HUVECs. That may be one of the mechanisms of antiatherosclerotic effect of DHEA.ATRA seems no positive effect on DHEA func-tion.