In situ isolation of nuclei or nuclear proteins from adherent cells: a simple, effective method with less cytoplasmic contamination

BACKGROUND: Isolation of nuclei or nuclear proteins is a prerequisite for western blot, nuclear proteome profiling, and other evaluations of nuclear proteins. Here, we developed a simple method for in situ isolation of nuclei or nuclear proteins by in situ removing the extranuclear part of adherent cells via a classical nonionic detergent triton X-100. RESULTS: First, the feasibility of our method was confirmed by confocal microscopy, atomic force microscopy, scanning electron microscopy, dynamic light scattering, immunofluorescence imaging, and time-lapse dynamic observation. Next, the optimal concentration range (approximately 0.1-1% for ~ 10 min) of triton X-100 and the optimal treatment time (< 30 min) of 0.1-1% Triton X-100 for our method were determined via western blotting of eight extra-/ intra-nuclear proteins. Subsequently, the effectiveness, sensitivity, and cytoplasmic contamination of our method were tested by investigating the levels of phosphorylated p65 (a NF-κB subunit) in the nuclei of endothelial or tumor cells treated with/without lipopolysaccharide (LPS) via western blotting and by comparing with a commercial nuclear protein extraction kit (a classical detergent-based method). The data show that compared with the commercial kit our method obtained a higher yield of total nuclear proteins, a higher pP65 level in both control and LPS groups, and much lower content of GAPDH (as a reference for cytoplasmic contamination) in nuclei. CONCLUSIONS: The in situ isolation of nuclei or nuclear proteins from adherent cells in this study is a simple, effective method with less cytoplasmic contamination. This method/strategy has the potential of improving the quality of downstream evaluations including western blotting and proteomic profiling.

Essential Oil from Alpiniae Zerumbet Fructus Alleviates High Glucose-induced HUVEC Injury via TXNIP Signaling Pathway / 中国实验方剂学杂志

Objective:To study the protective effect of essential oil from Alpiniae Zerumbet Fructus （EOAZF） against high glucose （HG）-induced injury of human umbilical vein endothelial cells （HUVECs） <italic>in vitro</italic>， so as to provide experimental evidence for the treatment of diabetes-induced cardiovascular diseases with EOAZF. Method:The cells were divided into the normal group， model group （25 mmol·L^-1 glucose）， positive control group （100 mg·L^-1 vitamin C）， and the low- （0.25 μg·L^-1）， medium- （1 μg·L^-1）， and high-dose （4 μg·L^-1） EOAZF groups. The HUVECs were damaged by HG. The secretion amounts of malondialdehyde （MDA）， nitric oxide （NO）， and endothelin-1 （ET-1） in HUVECs of different groups were measured to assess the protective effect of EOAZF against HG-induced injury. The effects of EOAZF on the apoptosis and reactive oxygen species （ROS） generation of HUVECs damaged by HG were detected by Annexin V-fluorescein isothiocyanate/propidium iodide （Annexin V-FITC/PI） staining and dichloro-dihydro-fluorescein diacetate （DCFH-DA） assay. The protein and mRNA expression levels of thioredoxin interacting protein （TXNIP） and thioredoxin 1 （Trx-1） were determined by Western blot and Real-time polymerase chain reaction （Real-time PCR）， followed by the measurement of total intracellular Trx-1 activity with insulin disulfide reduction method. Result:The comparison with the control group revealed that the proliferation of HUVECs in the model group was significantly inhibited and their shape was damaged. Compared with the model group， EOAZF protected HUVECs against HG-induced injury in a concentration-dependent manner. The secretion amounts of MDA and ET-1 （<italic>P</italic><0.05） in the model group were increased in contrast to those in the control group， while the NO level was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at all the three concentrations， especially at 4 μg·L^-1， obviously reduced the secretion of MDA and ET-1 （<italic>P</italic><0.05）， but elevated NO after HG induction （<italic>P</italic><0.05）. The cell apoptosis assay and ROS detection results demonstrated that the apoptosis and ROS level in the model group were higher than those in the control group （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly lowered the ROS level and apoptosis （<italic>P</italic><0.05） of HUVECs damaged by HG. The Western blot assay and Trx-1 activity detection uncovered that the protein and mRNA expression levels of TXNIP in the model group were significantly up-regulated as compared with those in the control group （<italic>P</italic><0.05）， whereas the Trx-1 activity was decreased （<italic>P</italic><0.01）. Compared with the model group， EOAZF at 4 μg·L^-1significantly down-regulated the mRNA and protein （<italic>P</italic><0.05） expression levels of TXNIP and enhanced the total Trx-1 activity （<italic>P</italic><0.05） in HUVECs， thus suppressing the oxidative stress. Conclusion:EOAZF exerts the protective effects against HG-induced injury in HUVECs by improving the endothelial function and reducing intracellular ROS and apoptosis. Its efficacy in anti-oxidative stress may be related to the down-regulation of mRNA and protein expression levels of TXNIP and the enhancement of Trx-1 activity.

Effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma Extract on HUVECs Senescence Mediated by Endothelial Microparticles / 中国实验方剂学杂志

Objective:To investigate the effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract on endothelial microparticles （EMPs）-induced vascular endothelial cell senescence， and explore the possible mechanism. Method:Human umbilical vein endothelial cells （HUVECs） were used as the research objects， and the aged model was established with 10-12 passages of replicative senescence cells. The experimental cells were divided into young group （2-4 passage cells）， aged group （10-12 passage cells）， only EMPs intervention group （extract EMPs produced by aged cells to intervene young cells） and low dose， middle dose and high dose drug intervention groups （200， 300， 400 mg·L^-1）. Senescence related <italic>β</italic>-galactosidase （SA-<italic>β</italic>-gal） staining and cell cycle propidium iodide （PI） staining were used to determine cell senescence. Cell counting kit-8 （CCK-8） assay was used to screen the drug concentration. EMPs were extracted by two-step centrifugation， EMPs labeled with phycoerythrin （PE） anti-human CD31 antibody or fluorescein isothiocyanate （FITC） annexin V were detected by flow cytometry， intracellular reactive oxygen species （ROS） were detected by 2'，7'- dichlorofluorescein diacetate （DCFDA） staining. Result:After treatment with the drug， SA-<italic>β</italic>-gal activity of the aged cells significantly decreased （<italic>P</italic><0.01）， the S phase arrest was restored （<italic>P</italic><0.01）， and the number of CD31⁺ EMPs and annexin V⁺ EMPs secreted by aged cells decreased （<italic>P</italic><0.05）. Compared with the young group， only EMPs intervention group could induce increased SA-<italic>β</italic>-gal activity and S phase arrest in young cells （<italic>P</italic><0.05，<italic>P</italic><0.01）. However， after intervention of EMPs and the drug， EMPs-mediated increase of SA-<italic>β</italic>-gal activity was significantly inhibited and S phase arrest was restored （<italic>P</italic><0.05）. The increase of intracellular ROS induced by EMPs was also significantly inhibited by the drug （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract can delay the senescence of vascular endothelial cells by influencing EMPs， and the mechanism may be related to the inhibition of increased intracellular ROS induced by EMPs.

Effects of leptin-modified human placenta-derived mesenchymal stem cells on angiogenic potential and peripheral inflammation of human umbilical vein endothelial cells (HUVECs) after X-ray radiation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-KB (NF-kB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-KB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.

Comparative transcriptomic analysis of vascular endothelial cells after hypoxia/re-oxygenation induction based on microarray technology / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Objective: To provide comprehensive data to understand mechanisms of vascular endothelial cell (VEC) response to hypoxia/re-oxygenation. Methods: Human umbilical vein endothelial cells (HUVECs) were employed to construct hypoxia/re-oxygenation-induced VEC transcriptome profiling. Cells incubated under 5% O2, 5% CO2, and 90% N2 for 3 h followed by 95% air and 5% CO2 for 1 h were used in the hypoxia/re-oxygenation group. Those incubated only under 95% air and 5% CO2 were used in the normoxia control group. Results: By using a well-established microarray chip consisting of 58 339 probes, the study identified 372 differentially expressed genes. While part of the genes are known to be VEC hypoxia/re-oxygenation-related, serving as a good control, a large number of genes related to VEC hypoxia/re-oxygenation were identified for the first time. Through bioinformatic analysis of these genes, we identified that multiple pathways were involved in the reaction. Subsequently, we applied real-time polymerase chain reaction (PCR) and western blot techniques to validate the microarray data. It was found that the expression of apoptosis-related proteins, like pleckstrin homology-like domain family A member 1 (PHLDA1), was also consistently up-regulated in the hypoxia/re-oxygenation group. STRING analysis found that significantly differentially expressed genes SLC38A3, SLC5A5, Lnc-SLC36A4-1, and Lnc-PLEKHJ1-1 may have physical or/and functional protein-protein interactions with PHLDA1. Conclusions: The data from this study have built a foundation to develop many hypotheses to further explore the hypoxia/re-oxygenation mechanisms, an area with great clinical significance for multiple diseases.

The Effect of Abnormal Blood Flow on Human Umbilical Vein Endothelial Cells / 医用生物力学

Objective To study the effects of abnormal blood flow on the secretion of ET-1/NO and the expression of the mRNA and the protein of ET-1, eNOS, VCAM-1, ICAM-1 and MCP-1 in human umbilical vein endothelial cells (HUVECs), so as to explore the mechanism of atherosclerosis （AS） caused by abnormal hemodynamics. MethodsThe HUVECs were divided into stress group, wall pressure group and normal group according to the different stress. The HUVECs were cultured under the corresponding stress for 24 hours and then collected. The secretion levels of NO and ET-1 were detected by enzyme method and radioimmunoassay method. The mRNA expression levels of eNOS and ET-1 were detected by qPCR. The expression levels of the mRNA and the protein of VCAM-1, ICAM-1, MCP-1 were detected by qPCR and Western blot. Results Compared with normal group, the secretion level and the mRNA expression level of ET-1 in wall pressure group increased significantly (P＜0.01), and the secretion level of NO and the mRNA expression level of eNOS in stress group also increased significantly (P＜0.01), The expressions level of the mRNA and the protein of VCAM-1, ICAM-1 and MCP-1 obviously increased in stress group and wall pressure group (P＜0.01). Conclusions Stress or wall pressure acting on HUVECs alone could lead to its dysfunction of the secretion and the expression of gene and protein. The mechanism of AS caused by abnormal blood flow was related to these dysfunction of HUVEC.

The Effect of Abnormal Blood Flow on Human Umbilical Vein Endothelial Cells / 医用生物力学

Objective To study the effects of abnormal blood flow on the secretion of ET-1/NO and the expression of the mRNA and the protein of ET-1, eNOS, VCAM-1, ICAM-1 and MCP-1 in human umbilical vein endothelial cells (HUVECs), so as to explore the mechanism of atherosclerosis （AS） caused by abnormal hemodynamics. MethodsThe HUVECs were divided into stress group, wall pressure group and normal group according to the different stress. The HUVECs were cultured under the corresponding stress for 24 hours and then collected. The secretion levels of NO and ET-1 were detected by enzyme method and radioimmunoassay method. The mRNA expression levels of eNOS and ET-1 were detected by qPCR. The expression levels of the mRNA and the protein of VCAM-1, ICAM-1, MCP-1 were detected by qPCR and Western blot. Results Compared with normal group, the secretion level and the mRNA expression level of ET-1 in wall pressure group increased significantly (P＜0.01), and the secretion level of NO and the mRNA expression level of eNOS in stress group also increased significantly (P＜0.01), The expressions level of the mRNA and the protein of VCAM-1, ICAM-1 and MCP-1 obviously increased in stress group and wall pressure group (P＜0.01). Conclusions Stress or wall pressure acting on HUVECs alone could lead to its dysfunction of the secretion and the expression of gene and protein. The mechanism of AS caused by abnormal blood flow was related to these dysfunction of HUVEC.

Effect of Periplaneta Aamericana Polypeptide on Angiogenesis / 中国实验方剂学杂志

Objective: To investigate the effect of the Periplaneta Americana polypeptide on the angiogenesis. Method:Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and cell scratch assay were used to observe effect of different concentration (6.25,12.5,25,50,100 mg·L-1) of the Periplaneta Americana polypeptide, CⅡ-3 and skimmed cream on the proliferation and migration of human umbilical vein endothelial cells (HUVECs), and a normal group and a thalidomide group were also established in this study. The tubule formation assay was used to detect the effect of different concentration (25, 50, 100 mg·L-1) of the Periplaneta Americana extracts on the formation of tubules in HUVECs cells. The adhesion between HepG2 cells and HUVECs cells was observed by cell adhesion assay. The expression of vascular endothelial growth factor (VEGF) proteins in HUVECs was detected by immunocytochemical staining and enzyme linked immunosorbent assay (ELISA). Result:MTT results showed that the Periplaneta Americana polypeptide could inhibit the proliferation of HUVECs in a dose-dependent manner (PPPPPPPPPConclusion:The Periplaneta Americana polypeptide can inhibit the invasion, metastasis and tube formation of HUVECs, and down-regulate the expression of VEGF in HUVECs. The effect of Periplaneta Americana polypeptide is better than CⅡ-3 and skimmed cream, and the among the polypeptide, the effect of PAP-2 is superior to the other two.

Serial MR Imaging of Magnetically Labeled Humen Umbilical Vein Endothelial Cells in Acute Renal Failure Rat Model

PURPOSE: To evaluate the usefulness of in vivo magnetic resonance (MR) imaging for tracking intravenously injected superparamagnetic iron oxide (SPIO)-labeled human umbilical vein endothelial cells (HUVECs) in an acute renal failure (ARF) rat model. MATERIALS AND METHODS: HUVECs were labeled with SPIO and poly-L-lysine (PLL) complex. Relaxation rates at 1.5-T MR, cell viability, and labeling stability were assessed. HUVECs were injected into the tail vein of ARF rats (labeled cells in 10 rats, unlabeled cells in 2 rats). Follow-up serial T2*-weighted gradient-echo MR imaging was performed at 1, 3, 5 and 7 days after injection, and the MR findings were compared with histologic findings. RESULTS: There was an average of 98.4+/-2.4% Prussian blue stain-positive cells after labeling with SPIO-PLL complex. Relaxation rates (R2*) of all cultured HUVECs at day 3 and 5 were not markedly decreased compared with that at day 1. The stability of SPIO in HUVECs was maintained during the proliferation of HUVECs in culture media. In the presence of left unilateral renal artery ischemia, T2*-weighted MR imaging performed 1 day after the intravenous injection of labeled HUVECs revealed a significant signal intensity (SI) loss exclusively in the left renal outer medulla regions, but not in the right kidney. The MR imaging findings at days 3, 5 and 7 after intravenous injection of HUVECs showed a SI loss in the outer medulla regions of the ischemically injured kidney, but the SI progressively recovered with time and the right kidney did not have a significant change in SI in the same period. Upon histologic analysis, the SI loss on MR images was correspondent to the presence of Prussian blue stained cells, primarily in the renal outer medulla. CONCLUSION: MR imaging appears to be useful for in vivo monitoring of intravenously injected SPIO-labeled HUVECs in an ischemically injured rat kidney.

Epigallocatechin-3-gallate Regulates Inducible Nitric Oxide Synthase Expression in Human Umbilical Vein Endothelial Cells / 한국실험동물학회지

Inducible nitric oxide synthase (iNOS) is a main enzyme producing nitric oxide during inflammation and thus contributes to the initiation and development of inflammatory cardiovascular diseases such as atherosclerosis. Epigallocatechin-3-gallate (EGCG), the major catechin derived from green tea, has multiple beneficial effects for treating cardiovascular disease, but the effect of EGCG on the expression of vascular iNOS remains unknown. In this study, we investigated (i) whether EGCG inhibits the expression of vascular iNOS induced by angiotensin II in human umbilical vein endothelial cells and, if it does inhibit, (ii) mechanisms underlying the inhibition. Angiotensin II increased expression levels of vascular iNOS; EGCG counteracted this effect. EGCG increased the production of reactive oxygen species. Moreover, EGCG did not affect the production of reactive oxygen species induced by angiotensin II. These data suggest a novel mechanism whereby EGCG provides direct vascular benefits for treating inflammatory cardiovascular diseases.

Effects of the knockdown of hypoxia inducible factor-1alpha expression by adenovirus-mediated shRNA on angiogenesis and tumor growth in hepatocellular carcinoma cell lines / 대한간학회지

BACKGROUND/AIMS: Hypoxia-inducible factor-1alpha (HIF-1alpha) is a central transcriptional factor involved in the cellular responses related to various aspects of cancer biology, including proliferation, survival, and angiogenesis, and the metabolism of the extracellular matrix in hypoxia. This study evaluated whether adenovirus-mediated small hairpin RNA (shRNA) against HIF-1alpha (shHIF-1alpha) inhibits cell proliferation and angiogenesis in hepatocellular carcinoma (HCC) cell lines. METHODS: Knockdown of HIF-1alpha expression was constructed by adenovirus-mediated RNA interference tools, and HCC cell lines infected with shHIF-1alpha coding virus were cultured under a hypoxia condition (1% O2) for 24 hours. Following infection, the expression levels of HIF-1alpha, angiogenesis factors, and matrix metalloproteinase (MMP) were examined using Western blotting. Cell proliferation and angiogenesis were measured by a cell proliferation assay (MTT assay) and an angiogenesis-related assay (invasion and tube-formation assay), respectively. RESULTS: Adenovirus mediated inhibition of HIF-1alpha induced suppression of tumor growth in HCC cell lines. It also down-regulated the expression of angiogenesis factor and MMP proteins. Angiogenesis as well as mobility of vascular cells to tumor was suppressed by adenovirus-mediated shHIF-1alpha-infected groups in human umbilical vein endothelial cells (HUVECs). CONCLUSIONS: These data suggest that adenovirus-mediated inhibition of HIF-1alpha inhibits the invasion, tube formation, and cell growth in HUVECs and HCC cells.

Effect of Jiuqiang Naoliqing-containing serum on secretion of nitric oxide and endothelin-1 in human umbilical vein endothelial cells / 中国康复理论与实践

@#ObjectiveTo investigate the effect of Jiuqiang Naoliqing(JNQ)-containing serum on secretion of nitric oxide(NO) and endothelin-1(ET-1) in human umbilical vein endothelial cells(HUVECs), in order to explore the effect of JNQ on regulation of vascular active factors in HUVECs.MethodsThe HUVECs were explored to different volume fractions of JNQ containing serum after being isolated and cultured. The levels of cultured medium of NO and ET-1 were measured.ResultsThe cultured medium content of NO and ET-1 in different volume fractions of JNQ containing serum was significantly increased compared with normal serum control (P<0.05), while the ratio of NO to ET-1 were increased in comparison with normal control (P<0.05).Conclusion JNQ can be promoted secretion of NO and ET-1 in HUVECs, preserving endothelial function, and maintaining the balance of NO/E-1.