Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (K_d=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent.     

内质网应激分子GRP78 对非小细胞肺癌的诊断价值研究

目的:探讨内质网应激分子葡萄糖调节蛋白78(GRP78)对非小细胞肺癌(NSCLC)的诊断价值。方法:收集45例NSCLC患者术后癌组织标本,及其18例癌旁组织,采用实时定量PCR法检测所有组织中GRP78的表达情况,分析NSCLC癌组织中GRP78的表达与患者临床特征的关系,及其对患者生存期的影响。结果:NSCLC组织中GRP78高表达率明显高于癌旁组织,差异有统计学意义(P0.05)。而Ⅲa期NSCLC患者癌组织中GRP78的表达水平明显高于Ⅰ-Ⅱ期,两者存在显著性的差异(P0.05)。Kaplan-Meier分析显示,GRP78高表达的患者其生存期明显低于GRP78低表达的患者(P0.05)。结论:NSCLC患者GRP78的表达可能与肿瘤细胞的发生、发展及患者的生存期有关,可作为一个预测NSCLC患者诊断及治疗的重要的分子标志物。     

血管内皮生长因子受体-2所介导信号通路的研究进展

血管新生是许多生理和病理进程发生的重要机理．在生物体内,血管新生需经过多步精细调控历程,现有研究表明,血管内皮生长因子(VEGF)及其受体蛋白酪氨酸激酶,尤其是血管内皮生长因子受体-2(VEGFR-2)所介导的信号级联通路是其中关键性的调节途径．VEGF/VEGFR-2所介导的信号级联通路可以调控血管内皮细胞的增殖、迁移、存活和通透性的改变,促进血管的新生．VEGF与VEGFR-2的胞外区特异性结合后,引起受体的二聚化和自身的交互磷酸化,使胞内特定的酪氨酸残基磷酸化．下游信号蛋白可以通过其Src同源结构域-2(SH2)与VEGFR-2结合,随后激活下游的效应蛋白,调控内皮细胞的生物学活性．此外,VEGF/VEGFR-2信号通路还可以下调树突细胞(DC)的活性．对VEGF/VEGFR-2信号通路作用的深入了解,将有助于新药的研发．     

Anti-KDEL-coated nanoparticles: A promising tumor targeting approach for ovarian cancer?

The purpose of this study was to target ovarian cancer cells by coupling paclitaxel (Tx)-loaded nanoparticles (NPs-Tx) to antibodies against KDEL sequence, able to recognize GRP94 and GRP78 that are located at cell surface in cancer cells whereas they are in the endoplasmic reticulum in healthy cells. Tx-loaded poly (dl-lactic acid) nanoparticles coated with anti-KDEL antibodies (NPs-Tx-KDEL) were successfully prepared and characterized. Interaction between tumor cells and NPs-Tx or NPs-Tx-KDEL was observed by microscopy with fluorescently labeled NPs and the efficacy of the different formulations was compared by a viability assay.     

拉西地平对内质网应激导致左室重塑的保护作用

目的:研究在高温高湿应激状态下拉西地平对葡萄糖调节蛋白(glucose-regulated protein78,GRP78)和C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(C/EBP-homologous protein,CHOP)在大鼠心肌中表达及对心室重塑的影响。方法:将30只雄性Sprague-Dawly(SD)大鼠随机分为对照组、高温高湿组、拉西地平干预组,每组10只。喂养6周后颈动脉插管测定平均动脉压及心率。B超检测左室形态结构。免疫组化法检测大鼠心肌GRP78及CHOP蛋白及表达水平。结果:高温高湿组的大鼠平均动脉压(MBP)、隔厚度(IVST)、左室后壁厚度(LPWT)、左室重量指数(LVWI),GRP78及CHOP蛋白表达水平与对照组相比均有显著升高(p<0.01),拉西地平干预组能显著降低大鼠平均动脉压(MBP)、室间隔厚度(IVST)、左室重量指数(LVWI),GRP78及CHOP蛋白的表达水平(p<0.05)。结论:内质网应激可能参与了高温高湿诱导的左室重构;拉西地平可能通过降低GRP78及CHOP的表达干预了ERS介导的心肌肥厚通路,从而改善心脏功能。     

拉西地平对高温高湿应激大鼠血管平滑肌细胞GRP78 和CHOP表达的影响

目的:探讨钙离子拮抗剂拉西地平对高温高湿应激大鼠血管平滑肌细胞内内质网应激相关因子葡萄糖调节蛋白78(glucose-regulated protein of 78kD,GRP78)和C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(CAAT/enhancer binding protein homologous protein,CHOP)表达的影响。方法:将60只雄性SD大鼠随机分为对照组、高温高湿组、拉西地平组,每组20只。按实验时间(2w、4w、6w、8w)的不同,各组又分为4个亚组,每个亚组5只大鼠。用颈动脉插管法测定各组大鼠的平均动脉压(MAP);用免疫组织化学法检测GRP78和CHOP的表达水平。结果:①高温高湿各组的MAP随着实验时间的延长呈逐渐递增的趋势,高温高湿4w、6w、8w亚组的MAP均显著高于相应的对照组和拉西地平组(P<0.05)。②随着实验时间的延长,高温高湿组GRP78表达量不断增加,6w达到最大值,8w表达减弱。高温高湿4w、6w、8w亚组GRP78表达量均高于相应对照组和拉西地平组,有显著性差异(P<0.05)。③高温高湿组2w、4w、6w、8w亚组CHOP表达量组间比较有显著性差异(P<0.05),8w亚组表达达到最高值;高温高湿组6w、8w亚组与相应对照组和拉西地平组比较有显著性差异(P<0.05)。结论:高温高湿应激可引起血管平滑肌细胞内质网应激反应,导致GRP78表达及CHOP表达的不对称增加,提示高温高湿应激可引起血管平滑肌细胞的损害;拉西地平可以减轻内质网应激,逆转高温高湿应激所致的血管平滑肌细胞的损伤作用,对血管平滑肌细胞有保护作用。     

综述: 血管衰老及其机制

血管老化是一个古老而又年轻的课题.本文综述了血管衰老的主要结构特征、功能改变及其机制的新近研究进展,重点就血管基质变化、内皮细胞衰老/功能失调、内皮祖细胞衰竭以及细胞间通讯等方面进行了阐述.     

衣霉素诱导大鼠心肌细胞内质网应激凋亡模型的构建

目的:通过衣霉素诱导内质网应激建立新生大鼠心肌细胞凋亡模型。方法:不同浓度、不同时间的衣霉素作用于原代培养乳鼠心肌细胞,通过MTT实验和流式细胞术测定心肌细胞的存活率和凋亡率,Western blot检测内质网应激蛋白GRP78,CHOP表达水平。结果:①与阴性对照组相比,衣霉素具有损伤心肌细胞的作用,并呈现剂量与时间依赖关系(P<0.05,n=12)。②通过流式细胞术判断心肌细胞死亡的性质,当衣霉素浓度为100ng/ml,作用72h时,心肌细胞存活率和凋亡率分别为57.4±3.2%(n=12),25.9±5.8%(n=3)。提示衣霉素损伤细胞的形式主要为凋亡性死亡。③内质网应激蛋白GRP78和CHOP表达于6h开始增加,24h达到峰值,随后呈下降趋势。结论:应用衣霉素成功诱导SD乳鼠心肌细胞内质网应激凋亡模型,衣霉素的最佳诱导浓度为100ng/ml,作用时间为72h。     

大肠癌LoVo细胞源外泌体通过传递pEGFR促进血管生成的研究

目的 探讨大肠癌LoVo细胞来源的外泌体（LoVo-Exos）对肿瘤血管生成的影响,并探讨其促血管生成的可能分子机制。方法 超速离心法提取LoVo-Exos,共聚焦显微镜观察其内化进入受体HUVEC细胞,体外管状形成实验分析LoVoExos对血管生成的影响;采用小鼠体内基质胶塞实验,分析LoVo-Exos对血管生成的影响。为探讨LoVo-Exos促血管生成的分子机制,蛋白质印迹（Western blot）分析LoVo-Exos携带磷酸化表皮生长因子受体（phosphorylated epidermal growth factor receptor,pEGFR）进入受体细胞。Western blot及酶联免疫吸附分析（ELISA）方法分析EGFR-ERK通路关键信号蛋白及下游血管生成核心分子表达情况,并观察敲减EGFR及细胞外调节蛋白激酶（extracellular regulatory protein kinase,ERK）抑制剂处理对血管生成的影响。结果 共聚焦显微镜观察到LoVo-Exos内化进入HUVEC内皮细胞;体外血管生成实验显示,LoVo-Exos能够显著促进HUVEC细胞管状结构的形成。小鼠皮下基质胶塞实验显示,LoVo-Exos能够促进血管样结构的形成。进一步研究发现,LoVo-Exos递送pEGFR到HUVEC中,激活EGFR-ERK通路,促进血管生成。分析LoVoExos对下游血管生成核心分子表达的影响,发现LoVo-Exos能够促进HUVEC细胞白介素-8(interleukin-8,IL-8)的分泌,敲减EGFR后LoVo-Exos中pEGFR水平降低,其对IL-8分泌的促进作用降低,且该促进作用能够被MEK1/2抑制剂U0126抑制。结论 LoVo-Exos能够在体内外促进血管生成,其可能机制为LoVo-Exos递送pEGFR到HUVEC中,通过EGFR-ERK途径上调IL-8分泌水平,从而促进HUVEC血管生成能力,为癌症的转移提供新机制。     

Enhanced autophagy blocks angiogenesis via degradation of gastrin-releasing peptide in neuroblastoma cells

Neuroblastoma is characterized by florid vascularization leading to rapid tumor dissemination to distant organs; angiogenesis contributes to tumor progression and poor clinical outcomes. We have previously demonstrated an increased expression of gastrin-releasing peptide (GRP) and its receptor, GRPR, in neuroblastoma and that GRP activates the PI3K-AKT pathway as a proangiogenic factor during tumor progression. Interestingly, AKT activation phosphorylates MTOR, a critical negative regulator of autophagy, a cellular process involved in the degradation of key proteins. We hypothesize that inhibition of GRPR enhances autophagy-mediated degradation of GRP and subsequent inhibition of angiogenesis in neuroblastoma. Here, we demonstrated a novel phenomenon where targeting GRPR using shRNA or a specific antagonist, RC-3095, decreased GRP secretion by neuroblastoma cells and tubule formation by endothelial cells in vitro. Furthermore, shGRPR or RC-3095 treatment enhanced expression of proautophagic proteins in human neuroblastoma cell lines, BE(2)-C, and BE(2)-M17. Interestingly, rapamycin, an inhibitor of MTOR, enhanced the expression of the autophagosomal marker LC3-II and GRP was localized within LC3-II-marked autophagosomes in vitro as well as in vivo, indicating autophagy-mediated degradation of GRP. Moreover, overexpression of ATG5 or BECN1 attenuated GRP secretion and tubule formation, whereas opposite effects were observed with siRNA silencing of ATG5 and BECN1. Our data supported the role of autophagy in the degradation of GRP and subsequent inhibition of angiogenesis. Therefore, activation of autophagy may lead to novel antivascular therapeutic strategies in the treatment of highly vascular neuroblastomas.     

CD157 Marks Tissue-Resident Endothelial Stem Cells with Homeostatic and Regenerative Properties

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Vascular permeability factor: a unique regulator of blood vessel function.

Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a potent polypeptide regulator of blood vessel function. VPF promotes an array of responses in endothelium, including hyperpermeability, endothelial cell growth, angiogenesis, and enhanced glucose transport. VPF regulates the expression of tissue factor and the glucose transporter. All of the endothelial cell responses to VPF are evidently mediated by high affinity cell surface receptors. Thus, endothelial cells have a unique and specific spectrum of responses to VPF. Since each of the responses of endothelial cells to VPF are also elicited by agonists, such as bFGF, TNF, histamine and others, it remains a major challenge to determine how post-receptor signalling pathways maintain both specificity and redundancy in cellular responses to various agonists.