Growth differentiation factor 9 inhibits vascular endothelial growth factor expression in human granulosa cells

Abstract

In aortic endothelial cells, the TGFβ signaling pathway is involved in the regulation of vascular endothelial growth factor (VEGF), which encodes a potent angiogenic factor crucial for the development of ovarian hyperstimulation syndrome. Growth differentiation factor 9 (GDF9) is a member of the TGFβ family and its effect on VEGF expression in human granulosa cells is unknown. In this study, human granulosa cells were collected from patients during the course of oocyte retrieval for in vitro fertilization and were cultured in vitro. After the first 48?h of culture, cells were treated with GDF9 with or without SB431542 (an ALK5 inhibitor) at various doses. The medium was then collected to determine the concentration of VEGF by ELISA. Cellular RNA was collected and extracted for quantification by real-time quantitative fluorescence PCR. Our study showed that GDF9 suppressed VEGF release from human granulosa cells in a dose-dependent manner and also downregulated VEGF mRNA levels in these cells. Furthermore, SB431542 antagonized the suppression of VEGF mRNA by GDF9 and diminished the inhibitory effect of GDF9 on VEGF release by human granulosa cells. Our results indicated that GDF9 can inhibit VEGF expression in human granulosa cells and ALK5 might mediate this process.     

N-乙酰半胱氨酸通过AMPK/SIRT1途径抑制缺氧诱导的大鼠脑血管内皮细胞损伤

目的研究N-乙酰半胱氨酸(NAC)对缺氧诱导脑血管内皮细胞损伤的调节作用及分子机制。方法选择SD大鼠分离培养脑血管内皮细胞,分为常氧组、缺氧组、0. 5 NAC组(缺氧+0. 5 mol/L NAC)、1. 0 NAC组(缺氧+1. 0 mol/L NAC)、NAC+8-bAMP组(缺氧+1. 0 mol/L NAC+1. 0 mol/L 8-bAMP)。采用MTS法检测细胞增殖活力,采用TUNEL染色检测凋亡率,采用试剂盒检测氧化应激指标,采用Western blot检测凋亡基因、AMPK/SIRT1通路分子的表达量。结果缺氧组细胞OD490值、T-AOC含量及B淋巴细胞瘤2(Bcl-2)、p-AMP活化蛋白激酶(pAMPK)、去乙酰化酶Sirtuin1(SIRT1)表达量均明显低于常氧组;缺氧组细胞凋亡率、细胞中活性氧(ROS)、丙二醛(MDA)、8-羟基脱氧鸟苷(8-OHDG)含量及bcl-2相关X蛋白(bax)、细胞色素C(Cyt-C)、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)的表达量均明显高于常氧组。0. 5 NAC组、1. 0 NAC组细胞OD490值、T-AOC量及Bcl-2、pAMPK、SIRT1表达量均明显高于缺氧组; 0. 5 NAC组、1. 0 NAC组细胞凋亡率、ROS、MDA、8-OHDG含量及Caspase-3、Cyt-C、Bax的表达量均明显低于缺氧组。NAC+8-bAMP组细胞OD490值、T-AOC及Bcl-2、p-AMPK、SIRT1表达量均明显低于1. 0 NAC组; NAC+8-bAMP组凋亡率、ROS、MDA、8-OHDG含量及Caspase-3、Cyt-C、Bax的表达量均明显高于1. 0 NAC组。结论 NAC能够通过激活AMPK/SIRT1通路来减轻氧化应激及线粒体凋亡介导的脑血管内皮细胞损伤。     

脑源性微囊泡对脐静脉内皮细胞骨架的影响研究

目的探讨脑源性微囊泡(BDMVs)对脐静脉内皮细胞骨架的影响。方法体外制备BDMVs,并予透射电镜观察及粒径检测。将PKH26荧光染料标记的BDMVs与脐静脉内皮细胞共培养0.5 h、1 h、2 h后,应用流式细胞术检测不同时间点的脐静脉内皮细胞吞噬BDMVs情况。将体外常规培养的脐静脉内皮细胞分为对照组、BDMVs组(加入终浓度1.5×10^7/mL的BDMVs处理细胞)及尼莫地平组[予2μg尼莫地平(0.2 mg/mL)预处理10 min后加入终浓度1.5×10^7/mL的BDMVs处理细胞],应用激光共聚焦显微镜观察罗丹明标记鬼笔环肽染色后各组脐静脉内皮细胞中纤维型肌动蛋白的荧光强度及应力纤维数目。结果透射电镜下可见体外制备的BDMVs具有完整的膜结构,粒径范围为100~1000 nm。流式细胞术检测显示,吞噬BDMVs的脐静脉内皮细胞比例随培养时间的延长呈时间依赖性升高(0.5 h:22.7%±1.2%;1 h:52.3%±1.3%;2 h:71.6%±1.9%),各时间点间两两比较差异均有统计学意义(P<0.05)。激光共聚焦显微镜观察显示,与对照组相比,BDMVs组中纤维型肌动蛋白的荧光强度明显升高且应力纤维数目明显增多增粗;而与BDMVs组相比,尼莫地平组中纤维型肌动蛋白的荧光强度明显降低且应力纤维数目明显减少变细。结论脐静脉内皮细胞吞噬BDMVs的作用随时间延长而增强,且吞噬BDMVs后可导致细胞骨架重构,而尼莫地平可部分阻断该作用。     

Toxicological study of metal and metal oxide nanoparticles in zebrafish

Metal and metal oxide nanoparticles have been widely used in catalytic, electronic and biomedical fields. It is necessary to investigate their toxicity and potential hazards to human and aquatic ecosystems. Zebrafish (Danio rerio), as a promising animal model, has been increasingly utilized to assess the toxicity of nanoparticles. Zebrafish has numerous characteristics for toxicity evaluation, such as short life cycle and high fecundity. This review describes the advantages of using zebrafish in the toxicity assessment of metal and metal oxide nanoparticles. Then we focus on the toxic effects, particularly the acute toxicity and the chronic ones, induced by nanoparticles in zebrafish. Target organ toxicities are also mentioned, including immunotoxicity, developmental toxicity, neurotoxicity, reproductive toxicity, cardiovascular toxicity and hepatotoxicity. The toxic effects of selected metal nanoparticles, including Au, Ag, Cu, and metal oxide nanoparticles such as TiO₂, Al₂O₃, CuO, NiO and ZnO, as well as the underlying mechanisms of nanoparticles causing these effects, are also highlighted and described in detail. Furthermore, we introduce the general factors that affect nanoparticle-induced toxicity in zebrafish. The drawbacks and advantages of using the zebrafish model in nanotoxicity studies are also argued. Finally, we suggest that the application of zebrafish to assess chronic toxicity of metal and metal oxide nanoparticles and the joint toxicity of metal and metal oxide nanoparticles and other pollutants could be hot topics in nanotoxicology.     

Exosomes from miRNA‐126‐modified endothelial progenitor cells alleviate brain injury and promote functional recovery after stroke

AimsWe previously showed that the protective effects of endothelial progenitor cells (EPCs)‐released exosomes (EPC‐EXs) on endothelium in diabetes. However, whether EPC‐EXs are protective in diabetic ischemic stroke is unknown. Here, we investigated the effects of EPC‐EXs on diabetic stroke mice and tested whether miR‐126 enriched EPC‐EXs (EPC‐EXs^miR126) have enhanced efficacy.MethodsThe db/db mice subjected to ischemic stroke were intravenously administrated with EPC‐EXs 2 hours after ischemic stroke. The infarct volume, cerebral microvascular density (MVD), cerebral blood flow (CBF), neurological function, angiogenesis and neurogenesis, and levels of cleaved caspase‐3, miR‐126, and VEGFR2 were measured on day 2 and 14.ResultsWe found that (a) injected EPC‐EXs merged with brain endothelial cells, neurons, astrocytes, and microglia in the peri‐infarct area; (b) EPC‐EXs^miR126 were more effective than EPC‐EXs in decreasing infarct size and increasing CBF and MVD, and in promoting angiogenesis and neurogenesis as well as neurological functional recovery; (c) These effects were accompanied with downregulated cleaved caspase‐3 on day 2 and vascular endothelial growth factor receptor 2 (VEGFR2) upregulation till day 14.ConclusionOur results indicate that enrichment of miR126 enhanced the therapeutic efficacy of EPC‐EXs on diabetic ischemic stroke by attenuating acute injury and promoting neurological function recovery.     

Nitric oxide donors offer protection to RBC from storage lesion

BackgroundRed blood cell (RBC), which is the most commonly transfused blood component, due to its ability to save a life in absence of any other blood components, can be stored up to maximum 6 weeks by following standard preservation procedure. During storage, RBC undergoes various biophysical and biochemical changes (commonly known as storage lesion) for which blood transfusion with “old RBC” shows a lot of clinical problems especially relevant to critically ill patients. Recent research on S-nitrosylation of haemoglobin to improve oxygen delivery of banked blood revealed the important role of nitric oxide (NO) in protecting storage lesion.Materials and methodsIn the present study, we used various “NO donating” chemicals with different NO release dynamics and chemistries in RBC storage cocktails to test the effects of NO on storage lesion. Changes in different storage markers were evaluated after 7 days storage of pre-treated RBC.ResultsAll the NO donors have shown protection against hemolysis. However, S-nitroso glutathione (GSNO) ranks first in shielding RBCs from storage lesion and additionally, it helps in elevating the value of 2, 3-di phosphoglycerate (2, 3-DPG), improving the RBC membrane fluidity and decreasing the adhesion towards endothelial monolayer.DiscussionPresent study reveals that NO released from NO donors confers protection against storage lesions of the RBC. Further, the study confirms that pre-treatment with GSNO, a NO donor and a nitrosylating agent, ensures the best protection to RBC during low temperature storage, when compared to other NO donor treatments.     

Poly-ICLC,a multi-functional immune modulator for treating cancer

Immunotherapies have become the first line of treatment for many cancer types. Unfortunately, only a small fraction of patients benefits from these therapies. This low rate of success can be attributed to 3 main barriers: 1) low frequency of anti-tumor specific T cells; 2) lack of infiltration of the anti-tumor specific T cells into the tumor parenchyma and 3) accumulation of highly suppressive cells in the tumor mass that inhibit the effector function of the anti-tumor specific T cells. Thus, the identification of immunomodulators that can increase the frequency and/or the infiltration of antitumor specific T cells while reducing the suppressive capacity of the tumor microenvironment is necessary to ensure the effectiveness of T cell immunotherapies.In this review, we discuss the potential of poly-ICLC as a multi-functional immune modulator for treating cancer and its impact on the 3 above mentioned barriers. We describe the unique capacity of poly-ICLC in stimulating 2 separate pattern recognition receptors, TLR3 and cytosolic MDA5 and the consequences of these activations on cytokines and chemokines production. We emphasize the role of poly-ICLC as an adjuvant in the setting of peptide-based cancer vaccines and in situ tumor vaccination by mimicking natural immune responses to infections. Finally, we summarize the impact of poly-ICLC in enhancing T infiltration into the tumor parenchyma and address the implication of this finding in the clinic.