Effects of cytokines on cultured microvascular endothelial cells derived from gerbil brain

Microvascular endothelial cells were isolated from gerbil brain and cultured. These cells retained an endothelial-specific marker, FVIII-related antigen. Alkaline phosphatase activity was also present in early passage. Two weeks after plating, these cells were attached to the culture dishes and had become like cobblestones in appearance. Then, the addition of tumor necrosis factor at a concentration of 1000 U/ml or more suppressed the DNA synthesis activity of endothelial cells by about 70% and induced morphological changes in the cells, which developed a spindle-like form and showed overlapping of cells, indicating loss of contact inhibition. The administration of interferon-tau induced no change. When a similar experiment was performed using culture supernatants of human glioma cells that had been cultured for a few days, DNA synthesis activity was suppressed by approximately 50% or more in 6 of 12 samples. The suppression of activity, however, was abolished by the addition of anti-tumor necrosis factor antibody in these 6 cases, suggesting the presence of activity resembling that of the tumor necrosis factor in the culture supernatants.     

Primary culture of microvascular endothelial cells from human benign prostatic hyperplasia

BACKGROUND: Prostate growth seems to be influenced by paracrine factors like IL-6 originating from the microvascular endothelium. Therefore, our efforts were focused on the primary culture and behavior of microvascular endothelial cells (HPEC) derived from tissue of human benign prostatic hyperplasia (BPH). Until now, the isolation and culture of HPEC from BPH have not been reported. METHODS: BPH tissue was cut into small cubes and gently squeezed after incubation with dispase. HPEC were cultured from the resulting cell suspension after a stepwise selection by use of superparamagnetic beads coated with antibodies against endothelial specific antigens. HPEC were characterized by flow cytometry and immunohistochemistry. gamma-Glutamyl transpeptidase activity (specific for microvascular endothelium) was measured after dissolution of the HPEC with Triton X-100. After the incubation of HPEC either with ATP, VEGF, or TNF-alpha, the release of IL-6 was measured by enzyme linked immunosorbent assay (ELISA). RESULTS: HPEC showed a typical endothelial morphology. They were positive for von Willebrand factor, CD31, CD62E (after stimulation with TNF-alpha), alpha-actin and were negative for fibroblastic antigens and PSA. Proliferation was stimulated by vascular endothelial growth factor (VEGF). gamma-Glutamyl transpeptidase activity in HPEC was 6.3 microIU/microg protein, whereas in human umbilical vein endothelial cells (HUVEC) no gamma-glutamyl transpeptidase activity was detectable. The IL-6 secretion of HPEC was stimulated by VEGF and TNF-alpha, but not by ATP and bradykinin. CONCLUSIONS: For the first time, the primary culture of microvascular endothelial cells from BPH tissue was successfully performed. Our results suggest that HPEC may be actively involved in prostate growth, due to the secretion of regulatory factors such as IL-6.     

罗哌卡因通过circ_0044516/微小RNA-198通路调控肺癌A549细胞增殖、迁移和侵袭的实验研究

目的:探讨罗哌卡因对肺癌A549细胞增殖、迁移和侵袭的影响及其机制。方法:2019年1月至2020年4月,将体外培养A549细胞(购自中国科学院上海细胞库)分为对照组、不同剂量[25、50、100 mg/L,罗哌卡因组(Rop组)]、乱序无意义阴性序列组(si-NC组)、si-circ_0044516组、Rop＋pcD...     

Trafficking of Shiga toxin/Shiga-like toxin-1 in human glomerular microvascular endothelial cells and human mesangial cells

This study has determined the intracellular transport route of Shiga-like toxin (Stx) and the highly related Shiga toxin in human glomerular microvascular endothelial cells (GMVECs) and mesangial cells. In addition, the effect of tumor necrosis factor-alpha (TNF-alpha), which contributes to the pathogenesis of hemolytic-uremic syndrome, was evaluated more profound. Establishing the transport route will provide better understanding of the cytotoxic effect of Stx on renal cells. For our studies, we used receptor-binding B-subunit (StxB), which is identical between Shiga toxin and Stx-1. The transport route of StxB was studied by immunofluorescence microscopy and biochemical assays that allow quantitative analysis of retrograde transport from plasma membrane to Golgi apparatus and endoplasmic reticulum (ER). In both cell types, StxB was detergent-resistant membrane associated and followed the retrograde route. TNF-alpha upregulated Gb3 expression in mesangial cells and GMVECs, without affecting the efficiency of StxB transport to the ER. In conclusion, our study shows that in human GMVECs and mesangial cells, StxB follows the retrograde route to the Golgi apparatus and the ER. TNF-alpha treatment increases the amount of cell-associated StxB, but not retrograde transport as such, making it likely that the strong TNF-alpha-induced sensitization of mesangial cells and GMVECs for the toxic action of Stx is not due to a direct effect on the intracellular trafficking of the toxin.     

糖皮质激素对人股骨头骨微血管内皮细胞功能的影响

[目的]通过基因芯片对人股骨头骨微血管内皮细胞表达谱分析及荧光定量PCR验证,探讨糖皮质激素对骨微血管内皮细胞损伤后发生的功能变化。[方法]采用本实验室建立的方法进行人股骨头骨微血管内皮细胞的分离、培养及鉴定,证明细胞为骨微血管内皮细胞。用0.1 mg/ml的含氢化可的松的培养基培养细胞,建立糖皮质激素骨微血管内皮细胞损伤模型。设正常对照组,用晶芯mRNA表达谱芯片对细胞损伤模型和对照组进行差异性转录本检测,对差异性表达基因行q PCR验证。[结果]mRNA芯片表达基因筛选结果发现ICAM-1、ET-1受体、PAI-1、血管紧张素II受体较对照组明显上调,e NOS、ET-1、PGI2合成酶、VEGF、PGE合成酶及PGE受体表达明显下调。q PCR验证结果与芯片结果一致。[结论]糖皮质激素促进了人股骨头骨微血管内皮细胞分泌缩血管因子、促凝血的因子及相关受体的表达,降低了舒张血管因子及相应受体的表达。     

Interleukin-6 protects LNCaP cells from apoptosis induced by androgen deprivation through the Stat3 pathway

BACKGROUND: Elevated expression of interleukin-6 (IL-6) is implicated in the progression of hormone refractory prostate cancer. Previous studies demonstrated that IL-6 promotes androgen-independent growth of prostate cancer cells. In this study, the effect of IL-6 on apoptosis induced by androgen deprivation was investigated. METHODS: The effect of IL-6 on apoptosis induced by androgen deprivation in LNCaP cells was examined by cell death ELISA and Western blot using cleaved poly (ADP-ribose) polymerase (PARP) and caspase-9, as well as Bcl-xL and phosphorylated Bad. The Stat3 in IL-6-mediated anti-apoptosis in prostate cancer cells was examined using either dominant-negative or constitutively activated Stat3 mutants. RESULTS: Overexpression of IL-6 renders androgen sensitive LNCaP human prostate cancer cells more resistant to apoptosis induced by androgen deprivation. LNCaP cells undergo apoptosis after 72 hr of androgen deprivation, an outcome is largely absent in clones overexpressing IL-6 as measured by cell death ELISA and chromatin degradation assays. IL-6 over-expressing cells resulted in a significant decrease in the expression of cleaved PARP and cleaved caspase-9 as well as an increase in the expression of Bcl-xL and phosphorylated Bad. Addition of IL-6 antibody completely abolished the anti-apoptotic activity of IL-6. This protective effect of IL-6 was reversed by the expression of a dominant-negative Stat3 mutant, Stat3F. Furthermore, ectopic expression of a constitutively active Stat3 antagonized androgen deprivation-induced cell death of LNCaP cells. CONCLUSION: These results indicate that IL-6 protects androgen sensitive LNCaP cells from apoptosis induced by androgen deprivation, and Stat3 activation play an important role in IL-6-mediated anti-apoptosis in prostate cancer cells.     

降钙素基因相关肽通过ERK1/2信号通路对人血管内皮细胞增殖的影响

[目的]探讨降钙素基因相关肽(CGRP)对人脐静脉血管内皮细胞(HUVECs)增殖的影响,并从ERK1/2信号通路角度初步探讨其可能的机制.[方法]实验分为4组,分别为空白对照组、CGRP组、CGRP+CGRP 8-37组、CGRP+PD98059组.AlarmarBlue法检测各组人脐静脉血管内皮细胞(HUVECs)的增殖变化情况;免疫印迹技术观察CGRP诱导后ERK1/2的磷酸化,及CGRP8-37、PD98059对ERK1/2磷酸化的影响.[结果](1) CGRP可诱导HUVECs细胞增殖,该作用可被CGRP8-37和PD98059阻断;(2) CGRP可时间依赖性地诱导HUVECs细胞ERK1/2的磷酸化,CGRP8-37和PD98059可减弱其作用.[结论]CGRP可诱导HUVECs细胞增殖,ERK1/2信号通路参与其调控机制.     

Ischemia postconditioning protects dermal microvascular endothelial cells of rabbit epigastric skin flaps against apoptosis via adenosine A2a receptors

Background: It has been shown that endogenous adenosine-induced by ischemia postconditioning attenuates apoptosis in recent studies; however, they focus only on parenchymal cells. The detailed mechanism has not been clearly clarified in any research and the subtype of adenosine receptors involved remains unknown. In our study, dermal microvascular endothelial cells (DMECs) are used to explore the role of adenosine A_2a receptor in the anti-apoptotic effects of ischemic postconditioning.

Material and methods: The epigastric skin flaps of rabbits were elevated. After 4?h of ischemia, the flaps were either abruptly reperfused or postconditioned by six cycles of brief reperfusion (15s) and re-ischemia (15s). Adenosine A2a receptor agonist (CGS-21680) and antagonist (ZM-241385) were used separately in other groups. The apoptosis-related proteins and adenosine A2a receptors were determined by immunohistochemical staining. Then apoptosis index was calculated by TUNEL.

Results: Ischemia/reperfusion caused severe damages in DMECs of flaps as demonstrated by an increase in apoptosis index and an increase in expressions of apoptosis-related proteins, which can be significantly attenuated by IPC treatment or exposure to a selective adenosine A_2a receptor agonist (all p values <.05). Meanwhile, the anti-apoptosis effects of IPC can be blocked by a selective adenosine A_2a receptor antagonist. Statistical analysis revealed that the increase of apoptosis index closely correlated inversely with the relative increase of adenosine A_2a receptors (p?Conclusions: Ischemia postconditioning protects DMECs of rabbit skin flap against apoptosis via activation of adenosine A_2a receptors.     

糖皮质激素通过PI3K-Akt-mTOR信号通路诱导股骨头骨微血管内皮细胞凋亡的研究

目的 探讨糖皮质激素通过PI3K-Akt-mTOR信号通路诱导股骨头骨微血管内皮细胞凋亡的机制。方法分离培养人骨微血管内皮细胞,取第3代细胞,给予不同剂量的氢化可的松处理24 h,浓度分别为0.1 mg/mL（低剂量）、0.2 mg/mL（中剂量）和0.3 mg/mL（高剂量）,Western blot法检测PI3K-Akt-mTOR信号通路关键蛋白磷脂酰肌醇激酶（PI3K）、蛋白激酶B(Akt)、哺乳动物雷帕霉素靶蛋白（mTOR）及其磷酸化形式的表达,AnnexinV/PI染色的流式细胞检测细胞凋亡。结果 Western blot电泳条带灰度值分析提示高浓度氢化可的松可以使该通路关键蛋白表达下降。与对照组相比,0.1 mg/mL、0.2 mg/mL和0.3 mg/mL三组的p-Akt表达分别下降60.72%、61.91%、100.00%（P＜0.01）。p-PI3K表达分别下降12.95%、36.26%、100.00%（P＜0.01）。p-mTOR表达分别下降22.95%、41.28%、57.11%（P＜0.01）。流式细胞检测显示正常对照组的早期凋亡、晚期凋亡/坏死以及活细胞比例分别为16.08%、30.86%和52.52%;激素损伤组这一比例分别为21.67%、37.2%、39.85%。激素损伤+PI3K抑制剂组这一比例分别为19.05%、37.9%和41.26%。统计学分析显示细胞比例组间差异均具有统计学意义（P＜0.001）。结论 糖皮质激素可以显著抑制人股骨头骨微血管内皮细胞内PI3K-Akt-mTOR信号通路关键蛋白的表达,从而诱导股骨头骨微血管内皮细胞发生凋亡和坏死。     

Comparison of two techniques to isolate microvascular endothelial cells from the omentum

The purpose of the study was to compare two different techniques for isolation of omental microvascular endothelial cells (ECs). Segments of unreinforced polytetrafluorethylene (PTFE) grafts, 9 cm long and 6 mm in diameter, were implanted in 22 dogs as an aortic interposition. Fourteen grafts were seeded with a mean of 7 x 10(5) viable ECs, derived from the microvessels of the omentum: eight grafts (group A) were seeded with ECs obtained by collagenase digestion and by filtration through a pore mesh; six grafts (group B) were seeded with ECs obtained by collagenase digestion and by Percoll gradient separation. In eight grafts (group C), the ECs were not added to the preclot mixture and served as a control. Animals were sacrificed 5 weeks after surgery. The percentage of thrombus-free area was 65 +/- 22% for group A grafts and 74 +/- 15% for group B grafts (NS). The subendothelial layer was 280 +/- 60 microns thick in group A and 220 +/- 30 microns thick in group B (P less than 0.05). Seeded grafts showed a higher production of 6-keto-PGF1 alpha after addition of sodium arachidonate than control grafts. Percoll gradient separation allows isolation of a more purified suspension of ECs. Refinements in omental EC procurement are still required to minimize contamination with other types of cells.     

可溶性酪氨酸激酶2融合蛋白对肿瘤坏死因子α诱导腹膜血管内皮细胞新生血管能力的影响

目的观察可溶性酪氨酸激酶2融合蛋白（sTie-2-Fc）对肿瘤坏死因子α（TNF-α）诱导腹膜血管内皮细胞新生血管能力的影响，及探讨TNF-α促进血管新生的机制。方法观察原代培养的人腹膜微血管内皮细胞经TNF-α作用后在基底膜基质（Matrigel）上形成管状结构的能力；在transwell板上迁移能力；对异硫氰酸荧光素标记的牛血清白蛋白（FITC-BSA）通透性的改变；以及用sTie-2-Fc干预后细胞上述功能的改变。结果TNF-α组与对照组相比，内皮细胞形成管状结构数显著增多[（70±7）个，4个视野比（17±4）个，4个视野，P〈0．05]；TNF-α＋sTie-2-Fc组[（40±6）个，4个视野]比TNF-α组管状结构数显著减少（P〈0．05）；sTie-2／Fc组和对照组间细胞管状结构数差异无统计学意义。TNF-α促进内皮细胞迁移能力可被sTie-2-Fc拮抗[（198±12）个／HP比（76±11）个／HP，P〈0．05]。对照组和sTie-2-Fc组间通透性差异无统计学意义；与对照组和sTie-2-Fc组比较，TNF-α组和TNF-α＋sTie-2-Fc组细胞通透性显著增加（P〈0．05），但两组间细胞通透性差异也无统计学意义。结论TNF-α促进腹膜血管新生与血管生成素及其受体有关。     

Pterostilbene protects against uraemia serum-induced endothelial cell damage via activation of Keap1/Nrf2/HO-1 signaling

International Urology and Nephrology - Chronic kidney disease causes uremia-related endothelial cell dysfunction associated with high risk for cardiovascular diseases. The vascular endothelium is...     

金黄色葡萄球菌和纤维连接蛋白结合蛋白A对血管内皮细胞紧密连接的破坏作用

目的观察金黄色葡萄球菌和纤维连接蛋白结合蛋白A基因（fnBA）敲除金黄色葡萄球菌菌株对人微血管内皮细胞（HMEC-1）紧密连接蛋白ZO-1、Claudin-5的影响,并探讨金黄色葡萄球菌侵袭血管内皮细胞的机制。 方法将野生金黄色葡萄球菌菌株NCTC8325与HMEC-1按100︰1比例共培养,实时定量RT-PCR检测共培养30 min、60 min和120 min时HMEC-1紧密连接成份ZO-1和Claudin-5 mRNA的表达,同时应用Western blot分析和免疫组织化学染色观察共培养不同时间紧密连接蛋白ZO-1和Claudin-5的表达。构建fnBA基因敲除突变菌株NCTC8325ΔfnbA,以野生金黄色葡萄球菌菌株NCTC8325为阳性对照,观察突变株NCTC8325ΔfnbA与HMEC-1共培养120 min后紧密连接蛋白ZO-1和Claudin-5的变化。 结果金黄色葡萄球菌NCTC8325与HMEC-1共培养30 min、60 min和120 min后紧密连接蛋白ZO-1、Claudin-5 mRNA的表达在30 min、120 min时较对照组显著下降[30 min时与对照组比较：ZO-1：t = 4.104、P = 0.015,Claudin-5 mRNA：t = 2.802、P = 0.049;120 min时与对照组比较：ZO-1：t = 3.478、P = 0.025,Claudin-5 mRNA：t = 1.802、P = 0.261],但60 min时ZO-1、Claudin-5 mRNA的表达有一过性升高。与金黄色葡萄球菌NCTC8325共培养后,免疫组织化学结果发现在30 min时ZO-1和Claudin-5两种紧密连接蛋白较对照组表达显著下降（t = 33.6、59.03,P均< 0.001）,120 min时ZO-1和Claudin-5两种紧密连接蛋白较对照组表达亦显著下降（t = 31.8、60.75,P均< 0.001）;Western blot与免疫组组织化学结果一致。与突变菌株NCTC8325ΔfnbA共培养30 min、60 min和120 min后,在30 min和60 min时ZO-1、Claudin-5蛋白的表达与NCTC8325组差异无统计学意义（P均> 0.05）,在120 min时ZO-1和Claudin-5蛋白的表达较NCTC8325组显著升高,差异均有统计学意义（ZO-1：t = 14.89、P < 0.001,Claudin-5：t = 7.008、P = 0.002）。 结论金黄色葡萄球菌能通过下调紧密连接蛋白破坏HMEC-1紧密连接屏障,且其表面蛋白FnBPA发挥了重要作用。     

PTGS-2-PTGER2/4 signaling pathway partially protects from diabetogenic toxicity of streptozotocin in mice

Prostanoids are suggested to participate in diabetes pathology, but their roles are controversially discussed. The purpose of the current study was to examine the role of cyclooxygenase (prostaglandin synthase [PTGS]) enzymes and prostaglandin (PG) E(2) signaling pathways in streptozotocin (STZ)-induced type 1 diabetes. Blood glucose, insulin, and survival rate were studied in mice with targeted disruption of the genes for PTGS and PGE receptors (PTGERs). PGE(2) was found as the main prostanoid formed by the pancreas. Contrarily to PTGS-1, deficiency of PTGS-2 activity significantly amplified STZ effect, causing dramatic loss of insulin production and rise in blood glucose and death rate. STZ metabolism was unaffected by PTGS deficiency. Diabetogenicity of STZ in PTGER1(-/-), PTGER2(-/-), PTGER3(-/-), and PTGER4(-/-) mice was comparable to control mice. In striking contrast, combined knockout of PTGER2 and PTGER4 by blocking PTGER4 in PTGER2(-/-) mice strongly enhanced STZ pathology. Treatment of PTGS-2(-/-) and wild-type mice with PTGER2/PTGER4 agonists partially protected against STZ-induced diabetes and restored β-cell function. Our data uncover a previously unrecognized protective role of PTGS-2-derived PGE(2) in STZ-induced diabetes mediated by the receptor types PTGER2 and PTGER4. These findings offer the possibility to intervene in early progression of type 1 diabetes by using PTGER-selective agonists.     

JAK2/STAT3信号通路在大脑缺血缺氧后小胶质细胞活化中的作用

缺血缺氧脑病(Hypoxic-ischemic encephalopathy,HIE)是导致新生儿死亡和婴幼儿神经发育障碍的主要原因,部分患儿有不同程度的神经系统后遗症,如脑瘫、认知和运动功能发育障碍。缺血缺氧可激活JAK2/STAT3信号通路,进而导致小胶质细胞异常活化,引发神经炎症反应;通过下调JAK2/STAT3信号通路可抑制小胶质细胞异常活化,改善神经系统炎性损伤。当前缺血缺氧脑病的治疗方法有限,因此研究小胶质细胞活化的调控机制对于缺血缺氧脑病的治疗具有重要临床价值。本文对JAK2/STAT3信号通路在小胶质细胞活化中的作用及二者相互关系的研究进展作一综述,以期为缺血缺氧脑病的治疗提供新的研究思路。     

Benzodiazepines safeguards nerve cells from the toxicity of lidocaine via miR-133a-3p/EGFR pathway

BackgroundLidocaine was an anesthetic commonly used for analgesia, but the neurotoxicity could not be ignored. However, benzodiazepines could alleviate the toxicity when combined with other drugs.PurposeTo explore the molecular mechanism of benzodiazepines in protecting nerve cells after the induction of lidocaine.MethodsPC12 cells were induced by lidocaine (0 mM, 0.1 mM, 0.5 mM and 1 mM) first and then treated by benzodiazepines (0 μM–200 μM). RT-qPCR assays measured RNA expressions of epidermal growth factor receptor (EGFR) and microRNA-133a-3p (miR-133a-3p) in PC12 cell line, respectively. Western blot was for protein detections of EGFR and caspase-3. Flow cytometry assay assessed apoptosis and cellular viability was validated via Cell Counting Kit-8 (CCK-8) test. Bioinformatics analysis predicted the potential link between miR-133a-3p and EGFR and the binding was verified using the Dual luciferase reporter experiment.ResultsBenzodiazepines increased cellular viability of PC12 cells up to 100 μM while suppressed viability between 100 and 200 μM. Benzodiazepines (0 μM, 10 μM, 50 μM and 100 μM) did not regulate PC12 cell viability but promoted the viability of lidocaine-treated PC12 cells. Lidocaine downregulated miR-133a-3p RNA expression but facilitated EGFR mRNA expression, which was reversed after treated by benzodiazepines. MiR-133a-3p targeted and negatively regulated EGFR expressions in mRNA and protein levels. Furthermore, miR-133a-3p inhibitor and overexpressed EGFR transfection both restrained the decreased PC12 cell viability and prompted cell apoptosis caused by benzodiazepines.ConclusionBenzodiazepines restrained lidocaine-induced toxicity in PC12 cells which secured viability and reduced apoptosis via miR-133a-3p/EGFR pathway.     

Activation of human microvascular endothelial cells with TNF-alpha and hypoxia/reoxygenation enhances NK-cell adhesion, but not NK-Cytotoxicity

BACKGROUND: Ischemia/reperfusion injury (I/R) and cellular rejection in solid organ transplantation are characterized by adhesion molecule up-regulation on the graft endothelium, a prerequisite for leukocyte recruitment. The contribution of NK cells to I/R and allograft rejection is not well understood. The aim of the present study was to investigate allogeneic interactions between human NK cells and microvascular endothelial cells (MVEC) with special regard to the differential impact of TNF-alpha and hypoxia/reoxygenation in an in vitro model of I/R. METHODS: MVEC were stimulated in vitro for 8 h with TNF-alpha, exposed to hypoxia (1% O2), hypoxia/reoxygenation, and combinations thereof in a hypoxia chamber. Cell surface expression of adhesion molecules on MVEC was analyzed by flow cytometry, and adhesion molecule shedding by ELISA. NK cell adhesion on MVEC was determined under shear stress, and NK cytotoxicity using Cr-release assays. RESULTS: Surface expression of ICAM-1, VCAM-1, and E-/P-selectin on MVEC was up-regulated by TNF-alpha but unaffected by hypoxia/reoxygenation in the absence of TNF-alpha. ICAM-1 expression was further increased by a combination of TNF-alpha and hypoxia/reoxygenation, whereas TNF-alpha-induced E-/P-selectin expression was strongly reversed by hypoxia/reoxygenation. NK cell adhesion increased after exposing MVEC to TNF-alpha and hypoxia/reoxygenation. Susceptibility of MVEC to NK cytotoxicity was enhanced by TNF-alpha and slighty reduced by hypoxia/reoxygenation. CONCLUSIONS: Endothelial activation with TNF-alpha, but not hypoxia/reoxygenation, induced NK cytotoxicity whereas the combination thereof induced the strongest NK cell adhesion. Our findings suggesting a role for NK cells in allograft responses support the development of anti-inflammatory treatment strategies to prevent I/R.