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Antiangiogenic Antithrombin Induces Global Changes in the Gene Expression Profile of Endothelial Cells

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Antiangiogenic Antithrombin Induces Global Changes in the Gene Expression Profile of Endothelial Cells Research Article Antiangiogenic Antithrombin Induces Global Changes in the Gene Expression Profile of Endothelial Cells Weiqing Zhang,1 Yung-Jen Chuang,3 Tianquan Jin,2 Richard Swanson,1 Yan Xiong,1 Lawrence Leung,3 and Steven T.Olson 1 1Center for Molecular Biology of Oral Diseases and 2Department of Oral Biology, College of Dentistry, University of Illinois at Chicago, Chicago, Illinois and 3Department of Medicine, Stanford University Medical Center, Stanford, California Abstract inhibitors. However, unregulated angiogenesis is observed under pathologic conditions such as tumor growth, diabetic retinopathy, Antithrombin, a serpin family protease inhibitor crucial to hemostasis, acquires antiangiogenic properties on undergoing and psoriasis. Angiogenesis plays an important role in tumor conformational alterations induced by limited proteolysis or growth, invasion, and metastasis (2). Compared with conventional elevated temperature. To better understand the biochemical clinical approaches for tumor therapy, antiangiogenesis-mediated mechanisms underlying antithrombin antiangiogenic activity, tumor therapy offers several unique advantages including de- we did genome-wide expression profiling, coupled with creased toxicity to the host, lack of drug resistance, and broad- quantitative reverse transcription-PCR, Northern blot, and spectrum efficacy against tumors of varied origins (3). Therefore, Western blot analyses, to characterize the gene expression using natural endogenous angiogenesis inhibitors to cut off the patterns that are induced by antiangiogenic antithrombin in nutrients necessary for cancer cell growth has become one of the cultured primary human umbilical vein endothelial cells. most promising tumor therapy approaches. Antithrombin is one of the most important endogenous Overall, 35 genes with significantly increased expression and regulators of blood coagulation (4). Recent studies have shown 93 genes with significantly reduced expression (z2-fold that conformationally altered cleaved and latent forms of changes) due to antiangiogenic antithrombin treatment were antithrombin have a strong antiangiogenic activity, as shown by identified. More than half of the down-regulated genes have their abilities to inhibit growth factor–stimulated proliferation, well-established proangiogenic functions in endothelial cells, migration, and capillary tube formation in cultured endothelial including cell-surface and matrix proteoglycans (e.g., perle- cells and to induce tumor regression in an in vivo mouse model can, biglycan, and syndecans 1 and 3) and mitogenesis-related (5–7). More interestingly, cleaved and latent forms of antithrombin signaling proteins (e.g., mitogen-activated protein kinase 3, were found to not only constitute major components of signal transducers and activators of transcription 2, 3, and 6, endogenous angiogenic inhibitors secreted by human primary and early growth response factor 1). In contrast, most up- pancreatic cancer cells, which were capable of blocking secondary regulated genes (e.g., caspase-3, p21, tissue inhibitor of tumor growth (8), but to also exhibit an inhibitory efficacy in metalloproteinases 1, 2, and 3, and adenomatosis polyposis human pancreatic cancer regression in mice comparable with coli) are known for their antiangiogenic functions which other well-known potent antiangiogenic agents such as endostatin include the promotion of cell apoptosis and cell cycle arrest and TNP-470 (9). Previous studies have shown that altered forms of and the inhibition of tumor growth and metastasis. These antithrombin exert their antiangiogenic effects by inducing cell results show that the antiangiogenic activity of antithrombin apoptosis (6, 9), inhibiting focal adhesion kinase activation (6), is mediated at least in part by a global genetic reprogram- causing cell cycle arrest (10), and down-regulating the expression ming of endothelial cells and strongly implicate an endothe- of the proangiogenic heparan sulfate proteoglycan (HSPG), lial cell ligand-receptor signaling mechanism in this perlecan, in endothelial cells (10). However, the precise biochem- reprogramming. (Cancer Res 2006; 66(10): 5047-55) ical mechanisms underlying antithrombin antiangiogenic activity still remain unclear. Because angiogenesis is a complex physiologic Introduction process which is tightly controlled by the integration of a Angiogenesis, the formation of new capillaries from preexisting multitude of gene activities (1), we expected that antithrombin vasculature by the proliferation, migration, and differentiation of would produce its antiangiogenic effects by inducing a unique endothelial cells, is a fundamental process required for a number antiangiogenic signaling network manifested by global alterations of physiologic and pathologic events (1). Under physiologic in endothelial cell gene expression. To test this hypothesis and conditions, angiogenesis is a highly regulated phenomenon and provide new insights into the biochemical mechanisms of is controlled by the balance between angiogenic stimulators and antithrombin antiangiogenic action, we sought to identify the gene expression pattern induced by antiangiogenic antithrombin treatment of endothelial cells using cDNA-based transcriptional profiling, coupled with real-time quantitative reverse transcriptase Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). PCR (RT-PCR), Northern blot, and Western blot analyses. This Current address for Y-J. Chuang: Institute of Bioinformatics and Structural Biology, effort led to the identification of 128 genes of which the expression National Tsing Hua University, Taiwan, Republic of China. z Requests for reprints: Weiqing Zhang, Center for Molecular Biology of Oral was altered 2-fold in antiangiogenic antithrombin–treated cells Diseases, University of Illinois at Chicago, Dentistry (M/C 860), 801 South Paulina relative to native antithrombin–treated and nontreated cells as Street, Chicago, IL 60612. Phone: 312-996-0652; Fax: 312-413-1604; E-mail: zhang98@ controls. Based on their functional similarities, 60% of the altered uic.edu. I2006 American Association for Cancer Research. genes were clustered into five major groups of genes of which the doi:10.1158/0008-5472.CAN-05-4449 activities are well-known regulators of angiogenesis. Our results www.aacrjournals.org 5047 Cancer Res 2006; 66: (10). May 15, 2006 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research show that antithrombin exerts its antiangiogenic effects in Quantitative RT-PCR analysis. Contaminating genomic DNA was cultured human endothelial cells at least in part by down- removed from isolated RNA by DNase I treatment (Ambion, Austin, TX). regulating the cluster of genes expressing (i)proangiogenic First-strand cDNA was reverse transcribed from total RNA using the SuperScript First Strand Synthesis System in the RT-PCR Kit (Invitrogen, heparan sulfate and other proteoglycans (e.g., perlecan, synde- A can-1, syndecan-3, biglycan, and proteoglycan 4), which serve as Carlsbad, CA) and was used at a final assay concentration of 0.1 ng/ L(ina 25 AL reaction volume). Real-time quantitation of mRNA was done in coreceptors for growth factors [basic fibroblast growth factor/ triplicate using a QuantiTect SYBR Green PCR kit and the ABI Prism vascular endothelial growth factor (bFGF/VEGF)] and other 7000HT Sequence Detection System (Applied Biosystems, Foster City, CA) ii cytokines, and ( ) mitogenic signaling molecules [e.g., early growth with 95% efficiency. In addition to profiling the mRNA target sequences in response factor 1, mitogen-activated protein (MAP) kinases, and cleaved antithrombin–treated, native antithrombin–treated, and untreated signal transducers and activators of transcription (STAT) family control endothelial cells, mRNAs of h-actin and glyceraldehyde-3-phos- proteins]. In addition, antiangiogenic antithrombin also induced phate dehydrogenase (GAPDH) were also profiled as controls. Forward and the expression of other genes [e.g., adenomatosis polyposis coli reverse primers (Supplementary Table S1) were designed based on previous (APC), caspase-3, TIMP family proteins, and p21] of which the publications or by using ABIs Primer Express software. For each single-well activities are associated with tumor suppression, induction of amplification reaction, a threshold cycle (Ct) was observed in the apoptotic death, inhibition of tumor cell migration, and cell cycle exponential phase of amplification and the quantitation of the test mRNA expression level was done relative to GAPDH mRNA levels measured in a arrest. Overall, our results provide an overview of an antiangio- separate reaction by determining the difference in threshold cycles between genic signaling network induced in cultured primary endothelial the test and GAPDH mRNAs (DCt = Cttest À CtGAPDH). The relative mRNA D cells by antithrombin, which contributes to the antiangiogenic level was then calculated from the formula (1.95) Ct, where the factor 1.95 activity of this serpin family protein. reflects the 95% amplification efficiency during each PCR cycle. Expression levels in antithrombin-treated cells were normalized to the levels measured Materials and Methods in untreated cells. Slot Northern blot analysis. cDNA clones encoding the various genes Cell culture and treatment. Fresh human umbilical cords were which were analyzed by slot Northern blots
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