Angiostatin enhances B7.1-mediated cancer immunotherapy independently of effects on vascular endothelial growth factor expression.

Tumors must develop an adequate vascular network to meet their increasing demands for nutrition and oxygen. Angiostatin, a multiple kringle (1-4)-containing fragment of plasminogen, is an effective natural inhibitor of tumor angiogenesis. Here we show that gene transfer of angiostatin into small (0.1 cm in diameter) solid EL-4 lymphomas established in syngeneic C57BL/6 mice led to reduced tumor angiogenesis and weak inhibition of tumor growth. In contrast, when angiostatin gene therapy was preceded by in situ gene transfer of the T-cell costimulator B7.1, large (0.4 cm in diameter) tumors were rapidly and completely eradicated, whereas B7.1 and angiostatin monotherapies were ineffective. Combined gene transfer of B7.1 and angiostatin generated potent systemic antitumor immunity that was effective in eradicating a systemic challenge of 10(7) EL-4 cells. Gene transfer of angiostatin expression plasmids led to overexpression of angiostatin in tumors, increased apoptosis of tumor cells, and decreased density of tumor blood vessels, which may allow the immune system to overcome tumor immune resistance. The latter effects were not the result of a decrease in vascular endothelial growth factor expression, as tumoral vascular endothelial growth factor expression increased slightly after angiostatin gene transfer, presumably in response to increasing hypoxia. These results suggest that combining immunogene therapy with a vascular attack by angiostatin is a particularly effective approach for eliciting antitumor immunity.     

Overexpression of vascular endothelial growth factor 165 (VEGF165) protects cardiomyocytes against doxorubicin-induced apoptosis

Doxorubicin (Dox) has been employed in cancer chemotherapy for a few decades. However its clinical application became restricted because of dose-dependent cardiomyopathy. Recent studies suggest that Dox-induced cardiomyocyte apoptosis is a primary cause of cardiac damage. Vascular endothelial growth factor (VEGF) is a major factor for endothelial cell survival and angiogenesis. We have previously shown that VEGF165 significantly attenuates oxidative stress-induced cardiomyocytes apoptosis. We hypothesized that VEGF165 will protect the cardiomyocytes from Dox-induced apoptosis. to evaluate our hypothesis, we transfected cardiomyocytes H9c2 with adenovirus expressing VEGF165 24 hours before the cells were challenged with Dox at a concentration of 2 μm. Cardiomyocyte apoptosis was evaluated by Annexin V-FITC staining and by Western blot detection of cleaved caspase-3. The hypothesis was confirmed, and the protective mechanisms involve the inhibition of death receptor-mediated apoptosis and up-regulation of the prosurvival Akt/Nf-κb/bcl-2 signaling pathway.     

A receptor for vascular endothelial growth factor that stimulates endothelial apoptosis.

Vascular endothelial growth factor (VEGF) is a dimeric angiogenic factor that is overexpressed by many tumors and stimulates tumor angiogenesis. VEGF initiates signaling by dimerizing the receptors VEGFR-1 and VEGFR-2. The Fas receptor stimulates apoptosis, and artificial dimerization of the Fas cytoplasmic domain has been shown to induce apoptosis. We constructed a chimeric receptor (VEGFR2Fas) combining the extracellular and transmembrane domains of VEGFR-2 with the cytoplasmic domain of Fas receptor. When VEGFR2Fas was stably expressed in endothelial cells in vitro, treatment with VEGF rapidly induced cell death with features characteristic of Fas-mediated apoptosis. These findings demonstrate that VEGFR2Fas functions as a VEGF-triggered death receptor and raise the possibility that introduction of VEGFR2Fas into tumor endothelium or tumor cells in vivo may convert tumor-derived VEGF from an angiogenic factor into an antiangiogenesis agent.     

X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells

Although the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to play an important role in the immunosurveillance of neoplasia, apoptotic factors that modulate the sensitivity of cancer cells to TRAIL are poorly understood. The inhibitor of apoptosis proteins (IAPs) have generated considerable interest as potential targets for cancer therapy, but the lack of a phenotype in X-linked IAP (XIAP) knockout mice has generated speculation that IAP function may be redundant. Using gene targeting technology, we show that disruption of the gene encoding XIAP in human cancer cells did not interfere with basal proliferation, but caused a remarkable sensitivity to TRAIL. These results demonstrate that XIAP is a nonredundant modulator of TRAIL-mediated apoptosis and provide a rationale for XIAP as a therapeutic target.     

Adenoviral modification of mouse brain derived endothelial cells, bEnd3, to induce apoptosis by vascular endothelial growth factor

A second generation genetically-engineered cell-based drug delivery system, referred to as apoptotic-induced drug delivery (AIDD), was developed using endothelial cells (ECs) that undergo apoptosis upon binding of vascular endothelial growth factor (VEGF) to a Flk-1:Fas fusion protein (FF). This new AIDD was redesigned using mouse brain derived ECs, bEnd3 cells, and an adenovirus vector in order to enhance and control the expression of FF. The FF was tagged with a HA epitope (FFHA) and designed to be coexpressed with green fluorescence protein (GFP) by the regulation of cytomegalovirus promoters in the adenovirus vector. bEnd3 cells showed favorable coexpression of FFHA and GFP consistent with the multiplicity of infection of the adenovirus. Immunofluorescence analysis demonstrated that FFHA was localized at the plasma membrane, whereas GFP was predominantly located in the cytoplasm of ECs. Cell death was induced by VEGF, but not by platelet derived growth factor or fibroblast growth factor in a dose-dependent manner (range 2-20 ng/ml), and revealed caspase-dependent apoptotic profiles. The FFHA expressing bEnd3 cells underwent apoptosis when cocultured with a glioma cell (SF188V+) line able to overexpress VEGF. The combined data indicated that the FFHA adenovirus system can induce apoptotic signaling in ECs in response to VEGF, and thus, is an instrumental modification to the development of AIDD.     

Activated endothelial cells induce apoptosis in lymphoma cells

We have previously shown that the arrest or regression of mouse liver metastases formed by lacZ-transduced ESbL T lymphoma cells (ESbL-lacZ) is associated with the stimulation of nitric oxide (NO) production by liver endothelial cells in sis. Here we studied in vitro the NO-mediated cytotoxicity against ESbL-lacZ target cells using well-characterized bovine endothelial cells (BEG) as effector cells. It was found that the co-culture of BEC with human TNF-alpha caused an increase of NO synthesis which could be completely blocked by the treatment with inducible NO synthase (iNOS) inhibitor N-G-monomethyl-L-arginine (NMMA). Incubation of activated BEC with metastatic lymphoma cells led to the death of the latter cells as evidenced by staining with propidium iodide and FAGS analysis. This cytotoxicity was considerably reduced after pretreatment of BEC with NMMA. Cytotoxic effects were also demonstrated after incubation of tumor cells with NO donor glycerol trinitrate (GTN). Non-activated BEC were not able to produce NO and showed a substantially lower level of cytotoxicity. The anti-tumor cytotoxicity exerted by activated BEC includes the stimulation of apoptosis in metastatic lymphoma cells which is mediated to a large extent by NO. These data reveal a novel role of endothelial cells in the elimination of metastatic cells through the induction of programmed cell death.     

Induced apoptosis in human prostate cancer cells by blocking of vascular endothelial growth factor by siRNA

Introduction

Vascular endothelial growth factor (VEGF) regulates several cell functions including; proliferation, differentiation, permeability, vascular tone, and the production of vasoactive molecules. The purpose of this study was to evaluate the potency of specific short-interfering RNA (siRNA) to suppress human VEGF expression by siRNA and investigate the effects of VEGF down-regulation on the cell proliferation and apoptosis of the human prostate cancer cell lines DU-145.

Methods

Transfection was performed using X-tremeGENE siRNA transfection reagent. At different time intervals, transfected cells were harvested and total RNA was extracted for RT-PCR. The VEGF content in supernatants were measured by ELISA. Inhibition of cell growth by hVEGF-siRNA was measured by using cell proliferation ELISA BrdU assay. Apoptotic cells were evaluated by using annexin-V-FITC apoptotic detection method.

Results

Transfection of hVEGF-siRNA resulted in statistically significant inhibition of hVEGF-mRNA that in turn caused a marked reduction in the expression of hVEGF. The cell growth was assessed every 24?h for 4?days after siRNA treatment resulted in a marked inhibition of cell proliferation as compared to scramble siRNA. The results of apoptosis showed that approximately 15?% of the cells treated with control-siRNA manifested evident apoptotic changes after 24?hpt, whereas DU-145 cells treated with hVEGF-siRNA significantly were positive, that is to say, 53?% at 72?hpt 23.9?±?2.78?% (P?<?0.001) and 13?±?1.57?% at 96?hpt.

Conclusion

Our findings indicate that siRNA are effective in eliciting the RNAi pathway in cancerous cells and that specific siRNA efficiently down-regulate VEGF expression. They could decrease VEGF production and induce apoptosis, which may also be linked to the inhibition of cancerous cell proliferation. Therefore, it can be concluded that siRNA-mediated suppression of VEGF represents a powerful tool against prostate cancer cell proliferation. VEGF down-regulation exerts a direct anti-apoptotic function in the DU-145 cell lines and promises the development of drugs for cancer therapy.     

慢病毒介导的Canstatin基因转移对脐静脉血管内皮细胞增殖与凋亡的影响

目的:构建携带Canstatin基因的慢病毒载体,体外转染脐静脉血管内皮细胞ECV304,观察其体外培养的脐静脉血管内皮细胞增殖及凋亡的影响。方法:应用基因重组技术构建慢病毒载体表达质粒pGC-FU-Canstatin,通过酶切、测序验证Canstatin基因后,将pGC-FU-Canstatin质粒和包装质粒pHelper 1.0,pHelper 2.0共同转染人胚胎肾上皮细胞系293T细胞,获得携带Canstatin基因重组慢病毒GC-FU-Cansta-tin,并测定病毒滴度。将重组慢病毒转染靶细胞人脐静脉血管内皮细胞ECV304,通过Western blotting检测靶细胞中Canstatin的表达。采用MTT法观察Canstatin基因对人脐静脉血管内皮细胞ECV304的体外细胞增殖的影响。TUNEL染色、流式细胞仪AnnexinV/碘化丙啶双染法,检测慢病毒介导的Canstatin基因诱导脐静脉血管内皮细胞的凋亡。结果:pGC-FU-Canstatin中携有正确的Canstatin基因序列;pGC-FU-Cansta-tin和包装质粒pHelper 1.0,pHelper 2.0共转染包装细胞293T产生重组病毒GC-FU-Canstatin;检测病毒滴度为1×109TU/ml;Western blotting检测到Canstatin蛋白在靶细胞中持续表达。Canstatin(感染复数分别为25和50)作用72h后,ECV304细胞增殖数目显著少于PBS组和空载体组(P<0.01);TUNEL染色,以MOI为25的重组慢病毒GC-FU-Canstatin作用于人脐静脉血管内皮细胞ECV304细胞72h后,出现典型的凋亡形态学改变。以感染复数为25的GC-FU-Canstatin处理ECV304细胞72h后,细胞凋亡率为(21.63±1.32)%,PBS组为(2.87±0.76)%,空载体组为(2.66±0.69)%,转基因组与空载体组、PBS组相比差异均有显著统计学意义(P<0.01)。结论:慢病毒介导的Canstatin基因可显著抑制人脐静脉血管内皮细胞ECV304的体外增殖,同时对人脐静脉血管内皮细胞的凋亡具有促诱导作用。     

肿瘤血管内皮细胞的免疫学特性

在肿瘤的发展过程中,肿瘤血管起到极为重要的作用,不仅为肿瘤提供营养、而且为肿瘤的转移提供了途径。由于肿瘤血管内皮细胞长期处于肿瘤微环境中,其表型及功能特性都发生了明显的变化,其中包括某些免疫学特性的改变,例如内皮细胞粘附分子表达水平降低、白细胞粘附作用减弱、抗原递呈功能不良、抗自由基损伤能力增强、合成大量的细胞外基质等。由于肿瘤血管内皮细胞是免疫细胞和免疫治疗药物进入肿瘤组织的第一道屏障,因此肿瘤血管内皮细胞的免疫学特性可能与肿瘤细胞逃避免疫监视、抵抗免疫杀伤有关。本文简要综述有关肿瘤血管内皮细胞免疫学特性,并分析这些特性与肿瘤免疫逃逸的关系。     

慢病毒介导的Canstatin基因转移对脐静脉血管内皮细胞增殖与凋亡的影响

目的：构建携带Canstatin基因的慢病毒载体,体外转染脐静脉血管内皮细胞ECV304,观察其体外培养的脐静脉血管内皮细胞增殖及凋亡的影响。方法：应用基因重组技术构建慢病毒载体表达质粒pGC-FU-Canstatin,通过酶切、测序验证Canstatin基因后,将pGC-FU-Canstatin质粒和包装质粒pHelper 1.0,pHelper 2.0共同转染人胚胎肾上皮细胞系293T细胞,获得携带Canstatin基因重组慢病毒GC-FU-Cansta-tin,并测定病毒滴度。将重组慢病毒转染靶细胞人脐静脉血管内皮细胞ECV304,通过Western blotting检测靶细胞中Canstatin的表达。采用MTT法观察Canstatin基因对人脐静脉血管内皮细胞ECV304的体外细胞增殖的影响。TUNEL染色、流式细胞仪AnnexinV/碘化丙啶双染法,检测慢病毒介导的Canstatin基因诱导脐静脉血管内皮细胞的凋亡。结果：pGC-FU-Canstatin中携有正确的Canstatin基因序列;pGC-FU-Cansta-tin和包装质粒pHelper 1.0,pHelper 2.0共转染包装细胞293T产生重组病毒GC-FU-Canstatin;检测病毒滴度为1×109TU/ml;Western blotting检测到Canstatin蛋白在靶细胞中持续表达。Canstatin（感染复数分别为25和50）作用72h后,ECV304细胞增殖数目显著少于PBS组和空载体组（P〈0.01）;TUNEL染色,以MOI为25的重组慢病毒GC-FU-Canstatin作用于人脐静脉血管内皮细胞ECV304细胞72h后,出现典型的凋亡形态学改变。以感染复数为25的GC-FU-Canstatin处理ECV304细胞72h后,细胞凋亡率为（21.63±1.32）%,PBS组为（2.87±0.76）%,空载体组为（2.66±0.69）%,转基因组与空载体组、PBS组相比差异均有显著统计学意义（P〈0.01）。结论：慢病毒介导的Canstatin基因可显著抑制人脐静脉血管内皮细胞ECV304的体外增殖,同时对人脐静脉血管内皮细胞的凋亡具有促诱导作用。     

Tetrapeptide DEVD-aldehyde or YVAD-chloromethylketone inhibits Fas/Apo-1 (CD95)-mediated apoptosis in renal-cell-cancer cells

The apoptotic machinery has been intensively investigated, and interleukin-l-beta-converting enzyme (ICE) and its homologs directly mediate apoptosis by means of their unique protease activity. Fas/Apol (CD95), a member of the TNF-receptor family, mediates apoptosis by binding to its ligand, which is mainly expressed on lymphocytes. Here, we investigated the expression and function of both molecules in renal-cell cancer (RCC). The expression of Fas was examined in 6 RCC cell lines by immunoblotting and all of them expressed Fas. ICE and CPP32/YAMA were also identified among the cell lines. We earlier examined ACHN cells expressing low levels of BCL-2, as well as KRC/Y cells with high levels of BCL-2. Here, we found that the anti-Fas monoclonal antibody, CH-11, induced apoptosis in a dose-dependent fashion more remarkably in ACHN cells. Pre-incubation with the tetrapeptide YVAD-chloromethylketone or DEVD-aldehyde inhibited Fas-mediated apoptosis. These findings suggest that, in RCC, apoptosis is induced by lymphocytes bearing Fas-L, and that it is achieved through the proteolytic action of CPP32/YAMA and/or ICE, or another member of the ICE/ced-3 protease family. © 1996 Wiley-Liss, Inc.     

Radiation-induced apoptosis in microvascular endothelial cells.

The response of the microvasculature to ionizing radiation is thought to be an important factor in the overall response of both normal tissues and tumours. It has recently been reported that basic fibroblast growth factor (bFGF), a potent mitogen for endothelial cells, protects large vessel endothelial cells from radiation-induced apoptosis in vitro. Microvessel cells are phenotypically distinct from large vessel cells. We studied the apoptotic response of confluent monolayers of capillary endothelial cells (ECs) to ionizing radiation and bFGF. Apoptosis was assessed by identifying changes in nuclear morphology, recording cell detachment rates and by detecting internucleosomal DNA fragmentation. Withdrawal of bFGF alone induces apoptosis in these monolayers. The magnitude of this apoptotic response depends upon the duration of bFGF withdrawal. Irradiation (2-10 Gy) induces apoptosis in a dose-dependent manner. Radiation-induced apoptosis occurs in a discrete wave 6-10 h after irradiation, and radiation-induced apoptosis is enhanced in cultures that are simultaneously deprived of bFGF. For example, 6 h after 10 Gy, 44.3% (s.e. 6.3%) of cells in the monolayer simultaneously deprived of bFGF exhibit apoptotic morphology compared with 19.8% (s.e. 3.8%) in the presence of bFGF. These studies show that either bFGF withdrawal or ionizing radiation can induce apoptosis in confluent monolayers of capillary endothelial cells and that radiation-induced apoptosis can be modified by the presence of bFGF.     

Negligible radiation protection of endothelial cells by vascular endothelial growth factor

Glioblastoma multiforme (GBM) is a radioresistant tumor. Tumor neoangiogenesis is an important mechanism for tumor sustenance. Angiogenesis is primarily mediated by vascular endothelial growth factor (VEGF), and earlier studies have suggested that VEGF protects human umbilical vein endothelial cells (HUVECs) against high doses of radiation. We tried to extend these findings to other endothelial cell lines and clinically relevant irradiation doses. Therefore, four different endothelial cell lines (HUVEC-C, primary HUVEC-P, an immortalized HUVEC cell line: EC-RF24, and bovine retina endothelial cells: BREC) were cultured without or with recombinant human VEGF165 (rhVEGF165). Cells were irradiated with gamma-rays from a 137Cs-source. Radiosensitivity was determined by proliferation or clonogenic assay. Apoptosis was assayed by flow cytometric determination of the sub-G1 population or by counting nuclear fragmentation. We found that the biologically active rhVEGF165 was able to improve clonogenic survival of HUVEC-C after 2 and 5 Gy. However, rhVEGF165 could not significantly alter the radiosensitivity of all cell lines studied in proliferation assays. rhVEGF165 only slightly reduced apoptosis in HUVEC-C after 3 Gy. In conclusion, the radioprotective effect from rhVEGF165 was found on different endothelial cell lines after clinically relevant radiation doses was negligible. We therefore hypothesize that the high VEGF-levels found in GBM in vivo do not reduce the radiosensitivity of endothelial cells, which is thought to contribute to the strong radioresistance of the tumor vasculature.     

Calcitriol (1,25-dihydroxycholecalciferol) selectively inhibits proliferation of freshly isolated tumor-derived endothelial cells and induces apoptosis

Calcitriol (1,25-dihydroxycholecalciferol) has antiproliferative and/or proapoptotic effects on many cell types and the glucocorticoid dexamethasone enhances these effects. We have shown that calcitriol modulates several key signaling proteins involved in differentiation, proliferation and apoptosis in tumor-derived murine endothelial cells (TDEC) and that these effects were not seen with endothelial cells isolated similarly from normal tissues. In the present study, TDEC and mouse embryonic yolk sac endothelial cells (MYSEC) were treated with calcitriol and followed over time for an effect. MYSEC were utilized as 'normal' control endothelial cells because they were more primitive, being isolated from a highly neovascular tissue, and had a similar morphology without the stimulus of the tumor microenvironment. The vitamin D receptor (VDR) is present in TDEC and MYSEC, and was upregulated in calcitriol-treated TDEC and MYSEC; dexamethasone further increased VDR expression following 48 h of treatment. The modulatory effects on signaling proteins were maximal by treatment for 48 h; phospho-Erk, phospho-Akt, p21 and bcl-2 were decreased in treated TDEC with the induction of p27 but there were no effects on MYSEC. After 48 h increased apoptosis was seen in treated TDEC by annexin V labeling with caspase-3 cleavage and decreased levels of poly(ADP-ribose) polymerase, but no effects were seen in MYSEC. Cell cycle analysis showed increased G(0)/G(1) arrest and an increase in the apoptotic sub-G(1) peak in treated TDEC but similar effects were not seen in MYSEC following 48-hour treatment. Proliferation assays were utilized and TDEC demonstrated decreased proliferation compared to normal endothelial cells at 48 h. To determine whether or not the VDR signaling was impaired in MYSEC, we performed the 24-hydroxylase (CYP24) promoter-luciferase reporter assay. CYP24 is a key enzyme involved in the breakdown of vitamin D. VDR signaling was intact in both cell types and calcitriol induced CYP24 mRNA expression in MYSEC but not in TDEC. Taken together, despite similar levels of VDR expression and intact signaling in both cell types, calcitriol selectively inhibits proliferation and induces apoptosis in TDEC with no effect on MYSEC. Thus calcitriol exerts differential effects on TDEC compared to normal cells.     

肿瘤血管内皮细胞异质性分子的研究进展

肿瘤血管与正常血管相比,其发生基础、表现形式及分子表达均存在明显差异,肿瘤血管内皮细胞的分子异质性是肿瘤抗血管生成靶向治疗的理论基础。深入研究肿瘤血管异质性分子对于肿瘤诊断、血管靶向治疗等具有重要意义。本文综述目前肿瘤血管内皮细胞异质性分子的研究进展,为相关研究提供参考。