Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones that overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knock down NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared with V189shCtl, under stress conditions. Unexpectedly, NRP1 knockdown had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity toward NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.Key words: VEGF isoforms, survival, apoptosis, NRP-1, breast cancer cells     

��v��3 Integrin Limits the Contribution of Neuropilin-1 to Vascular Endothelial Growth Factor-induced Angiogenesis

Both vascular endothelial growth factor receptors (VEGFR) and integrins are major regulators of VEGF-induced angiogenesis. Previous work has shown that β3 integrin can regulate negatively VEGFR2 expression. Here we show that β3 integrin can regulate negatively VEGF-mediated angiogenesis by limiting the interaction of the co-receptor NRP1 (neuropilin-1) with VEGFR2. In the presence of αvβ3 integrin, NRP1 contributed minimally to VEGF-induced angiogenic processes in vivo, ex vivo, and in vitro. Conversely, when β3 integrin expression is absent or low or its function is blocked with RGD-mimetic inhibitors, VEGF-mediated responses became NRP1-dependent. Indeed, combined inhibition of β3 integrin and NRP1 decreased VEGF-mediated angiogenic responses further than individual inhibition of these receptors. We also show that αvβ3 integrin can associate with NRP1 in a VEGF-dependent fashion. Our data suggest that β3 integrin may, in part, negatively regulate VEGF signaling by sequestering NRP1 and preventing it from interacting with VEGFR2.     

Neuropilins

It is now well established that neuropilins (NRP1 and NRP2), first described as mediators of neuronal guidance, are also mediators of angiogenesis and tumor progression. NRPs are receptors for the class-3 semaphorin (SEMA) family of axon guidance molecules and also for the vascular endothelial growth factor (VEGF) family of angiogenic factors. VEGF-NRP interactions promote developmental angiogenesis as shown in mouse knockout and zebrafish knockdown studies. There is also evidence that NRPs mediate tumor progression. For example, overexpression of NRP1 enhances tumor growth whereas NRP1 antagonists, such as soluble NRP1 and anti-NRP1 antibodies, inhibit tumor growth. Furthermore, some class-3 SEMAs acting via NRPs inhibit tumor angiogenesis, progression and metastasis. Clinical data suggest that high NRP levels correlate with poor prognosis and survival in a variety of cancer types. Taken together, these results suggest that NRPs are potentially valuable targets for new anti-cancer therapies. We analyze here the current knowledge on NRPs and their role in angiogenesis and tumor progression and enumerate strategies for targeting these receptors.     

Neuropilins: Novel Targets for Anti-Angiogenesis Therapies

It is now well-established that neuropilins (NRP1 and NRP2), first described as mediators of neuronal guidance, are also mediators of angiogenesis and tumor progression. NRPs are receptors for the class-3 semaphorin (SEMA) family of axon guidance molecules and also for the vascular endothelial growth factor (VEGF) family of angiogenic factors. VEGF-NRP interactions promote developmental angiogenesis as shown in mouse knockout and zebrafish knockdown studies. There is also evidence that NRPs mediate tumor progression. For example, overexpression of NRP1 enhances tumor growth whereas NRP1 antagonists, such as soluble NRP1 and anti-NRP1 antibodies, inhibit tumor growth. Furthermore, some class-3 SEMAs acting via NRPs inhibit tumor angiogenesis, progression and metastasis. Clinical data suggest that high NRP levels correlate with poor prognosis and survival in a variety of cancer types. Taken together, these results suggest that NRPs are potentially valuable targets for new anti-cancer therapies. We analyze here the current knowledge on NRPs and their role in angiogenesis and tumor progression and enumerate strategies for targeting these receptors.     

Properties of gene knockdown system by vector‐based siRNA in zebrafish

RNA interference (RNAi) has emerged as a powerful tool to silence specific genes. Vector‐based RNAi systems have been developed to downregulate targeted genes in a spatially and temporally regulated fashion both in vitro and in vivo. The zebrafish (Danio rerio) is a model animal that has been examined based on a wide variety of biological techniques, including embryonic manipulations, forward and reverse genetics, and molecular biology. However, a heritable and tissue‐specific knockdown of gene expression has not yet been developed in zebrafish. We examined two types of vector, which produce small interfering RNA (siRNA), the direct effector in RNAi system; microRNA (miRNA) process mimicking vectors with a promoter for RNA polymerase II and short hairpin RNA (shRNA) expressing vector through a promoter for RNA polymerase III. Though gene‐silencing phenotypes were not observed in the miRNA process mimicking vectors, the transgenic embryos of the second vector (Tg(zU6‐shGFP)), shRNA expressing vector for enhanced green fluorescence protein, revealed knockdown of the targeted gene. Interestingly, only the embryos from Tg(zU6‐shGFP) female but not from the male fish showed the downregulation. Comparison of the quantity of siRNA produced by each vector indicates that the vectors tested here induced siRNA, but at low levels barely sufficient to silence the targeted gene.     

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.     

miR-126 regulates angiogenic signaling and vascular integrity

Precise regulation of the formation, maintenance, and remodeling of the vasculature is required for normal development, tissue response to injury, and tumor progression. How specific microRNAs intersect with and modulate angiogenic signaling cascades is unknown. Here, we identified microRNAs that were enriched in endothelial cells derived from mouse embryonic stem (ES) cells and in developing mouse embryos. We found that miR-126 regulated the response of endothelial cells to VEGF. Additionally, knockdown of miR-126 in zebrafish resulted in loss of vascular integrity and hemorrhage during embryonic development. miR-126 functioned in part by directly repressing negative regulators of the VEGF pathway, including the Sprouty-related protein SPRED1 and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-beta). Increased expression of Spred1 or inhibition of VEGF signaling in zebrafish resulted in defects similar to miR-126 knockdown. These findings illustrate that a single miRNA can regulate vascular integrity and angiogenesis, providing a new target for modulating vascular formation and function.     

Cloning and functional analysis of sheep U6 promoters

Gene silencing mediated by small interfering RNA has become a powerful biological tool for the regulation of gene expression. In order to develop an effective short hairpin RNA (shRNA) expression vector, specifically for use in sheep species, we have identified two sheep U6 promoters based on the highly conserved polymerase III promoter elements. Promoter activity was measured by U6 promoter-driven shRNA to suppress enhanced green fluorescent protein (EGFP) expression. The knock down assay demonstrated that the two sheep U6 promoters and mouse U6 promoter induced a similar level of EGFP knockdown. These results suggest that the two sheep U6 promoters could efficiently drive shRNA expression for gene silencing and may have applications in RNAi-based sheep research.     

A miR-21 hairpin structure-based gene knockdown vector

RNA interference (RNAi) is widely used to study gene functions as a reverse genetic means from first-generation siRNA to second-generation short hairpin RNA (shRNA) or the newly developed microRNA (shRNA-miR). Here we report a gene knockdown vector system based on the mouse miR-21 hairpin structure. In this system, the pre-miRNA hairpin of the miR-21 gene was modified by replacing the 22-nucleotide mature sequence with shRNA sequences that target genes of interest, flanked by 160-bp upstream and 65-bp downstream sequences of the mouse pre-miR-21. We tested this system by knocking down the enhanced green fluorescence protein (EGFP) reporter gene using different vectors, in which shRNA-miR was driven by the polymerase II (pol II) promoter. We found that miR-21 hairpin-based shRNA-miR can be directly placed under pol II promoter, like UbC or CMV promoter to knockdown the gene of interest. To facilitate the wide application of the miR-21 hairpin-based gene knockdown system, we further knocked down the endogenous gene lamin (A/C), which showed that endogenous lamin A/C expression can be efficiently silenced using the miR-21 hairpin-based lentiviral vector. The miR-21 hairpin-based gene knockdown vector will provide a new genetic tool for gene functional studies in vitro and in vivo.     

Proteomic studies of rat tibialis anterior muscle during postnatal growth and development

Phosphoinositide 3-kinase (PI3K) pathway exerts its effects through Akt, its downstream target molecule, and thereby regulates various cell functions including cell proliferation, cell transformation, apoptosis, tumor growth, and angiogenesis. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been implicated in regulating cell survival signaling through the PI3K/Akt pathway. However, the mechanism by PI3K/PTEN signaling regulates angiogenesis and tumor growth in vivo remains to be elucidated. Vascular endothelial growth factor (VEGF) plays a pivotal role in tumor angiogenesis. The effect of PTEN on VEGF-mediated signal in pancreatic cancer is unknown. This study aimed to determine the effect of PTEN on both the expression of VEGF and angiogenesis. Toward that end, we used the siRNA knockdown method to specifically define the role of PTEN in the expression of VEGF and angiogenesis. We found that siRNA-mediated inhibition of PTEN gene expression in pancreatic cancer cells increase their VEGF secretion, up-modulated the proliferation, and migration of co-cultured vascular endothelial cell and enhanced tubule formation by HUVEC. In addition, PTEN modulated VEGF-mediated signaling and affected tumor angiogenesis through PI3K/Akt/VEGF/eNOS pathway.     

斑马鱼VEGF基因的siRNA表达载体构建、有效序列筛选及其对基因表达的干预性研究

为探索小干扰RNA（small interfering RNA,siRNA）表达质粒在研究斑马鱼血管内皮生长因子（vascular endothelial growth factor,VEGF）基因调控网络中的应用,构建了4个以斑马鱼VEGF基因为靶点的siRNA表达载体pSI—VEGF、pS2-VEGF、pS3-VEGF及pS4-VEGF。通过显微注射的方法将载体导入1-2细胞期斑马鱼体内,于胚胎发育的48h采用RT-PCR的方法检测VEGF基因的表达量,研究不同干扰序列对VEGF基因表达的干涉作用。结果显示,成功地构建了siRNA表达载体。针对不同位点的寡核苷酸序列抑制VEGF基因表达的效率有显著差异,其中注射了ps1-VEGF的胚胎出现了心包膜水肿、血流速度减慢、循环红细胞堆积等症状,同时肠下静脉、节间血管以及其它血管出现不同程度的发育缺陷。实验结果说明,pS1-VEGF可引起斑马鱼胚胎血管发育缺陷。