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

Research Article

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 were purchased from American obtained from normal-term deliveries at Stanford University Hospital. Type Culture Collection (Manassas, VA) or, in the case of p21WAF1/Cip1, Primary endothelial cells were isolated from umbilical cords as previously provided by Dr. Guy Adami (University of Illinois at Chicago, Chicago, IL). described (11) with modifications (10). Seventy-five percent confluent DIG-labeled probes for each selected gene in Northern-blot analyses were passage-2 human umbilical vein endothelial cells (HUVEC) in Falcon T12.5 synthesized by employing a DIG-Primer Labeling PCR kit from Roche flasks were treated with native and cleaved forms of human plasma Applied Science (Indianapolis, IN) along with the primers listed in A antithrombin (30 g/mL) in the presence or absence of bFGF (10 ng/mL; Supplementary Table S1 and the cDNA clones as template. Total RNA R&D Systems, Minneapolis, MN) for 24 hours with 0.5% heat-inactivated (2 Ag) prepared from antithrombin-treated or control HUVECs after fetal bovine serum and 1% antibiotics. These cells were used for microarray 24 hours was applied to a nylon membrane and hybridization and washing and real-time RT-PCR experiments. For Northern blot and Western blot were done under high-stringency conditions according to the protocol of experiments, primary HUVECs were obtained from Clonetics (San Diego, the manufacturer for the DIG-labeled slot Northern blot analysis system CA) and cells before passage 10 were treated with antithrombin as (Roche Applied Science). GAPDH mRNA levels were determined simulta- indicated for the microarray experiments. neously in each sample as an internal loading control. Native and cleaved forms of antithrombin. Human antithrombin was Western blot analysis. Preparation of endothelial cell lysates and purified from outdated plasma as described (12). The reactive loop cleaved Western blot analyses were done using procedures previously described form of antithrombin was obtained by treatment with human neutrophil (7, 10). Lysates from treated or untreated endothelial cells (50 Ag total elastase (Athens Research and Technology, Athens, GA) followed by removal protein) were electrophoresed in a 4% to 12% gradient SDS/polyacrylamide of the elastase by heparin-agarose chromatography as described (10). gel and transferred to an Immobilon P membrane. After blocking nonspecific RNA preparation and cDNA microarray analysis. RNA isolation from binding sites, blots were incubated with appropriate antibodies [all from treated and untreated endothelial cells and cDNA microarray analysis were Santa Cruz Biotechnology (Santa Cruz, CA) except for perlecan (Zymed done essentially as previously described (10). The only modification was Laboratories, San Francisco, CA) and h-actin (Sigma, St. Louis, MO)]. Blots that genes that were at least 2.0-fold induced or 2.0-fold repressed in were then exposed to horseradish peroxidase–conjugated secondary anti- cleaved antithrombin–treated cells compared with native antithrombin– bodies and visualized by the enhanced chemiluminescence system from treated cells in more than one of three microarray chip replicates were Amersham (Piscataway, NJ). considered to be significantly altered in their expression. After hybridiza- Morphologic detection of cellular apoptosis. Sixty to seventy percent tion with Cy3-labeled test cDNA and Cy5-labeled reference cDNA, the confluent HUVECs in 12-well plates were incubated for 24 hours with fluorescence signals from each gene array chip were scanned. The data 30 Ag/mL of antithrombin in 0.5% bovine calf serum-F-12K (Invitrogen). were retrieved as Cy3/Cy5 fluorescence intensity ratios after normalization Cells were harvested and resuspended in PBS containing 5% glycerol and by setting the average log fluorescence ratio for all array elements on the 0.1 mol/L NaCl. The cells were dried onto slides, fixed with acetone/ chip equal to zero using the Stanford Microarray Database software (13). methanol (1:1), and the cell nuclei were stained with 4¶,6-diamidino- For UniGene clusters represented by multiple arrayed elements, the mean 2-phenylindole (DAPI; 500 ng/mL). Apoptotic cells appearing as fractured fluorescence ratios (for all elements representing the same UniGene nuclei were counted in random fields by fluorescence microscopy (Â200 cluster) were reported. Genes of which the expression was significantly magnification, at least 10 fields per sample) and photographed. altered in cleaved antithrombin–treated cells relative to native antithrombin–treated cells based on the selection criterion were filtered and the normalized fluorescence ratios observed for these genes in multiple chips Results were averaged for antithrombin-treated and untreated cell conditions. Genes were manually grouped by their functions into gene clusters using Genome-wide expression profiling. To identify target genes of the human gene database.4 which the expression is altered by antiangiogenic antithrombin treatment of endothelial cells, we profiled the expression of genes in primary HUVECs after 24-hour treatment with 30 Ag/mL of native antithrombin, cleaved antithrombin, or buffer alone using 4 http://pevsnerlab.kennedykrieger.org/dragon.htm. cDNA-based microarray analysis. Expression levels in cells treated

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Antiangiogenic Antithrombin Gene Expression Profiling with antiangiogenically active cleaved antithrombin which differed angiogenesis. For example, vitronectin has been shown to be a from the expression levels in cells treated with the antiangiogeni- necessary matrix molecule for mediating antithrombin antiangio- cally inactive native form of antithrombin by a factor z2.0 in at genic function (14). p21WAF1/Cip1 is well known for its cell cycle least two of three replicate microarray chips were considered to arrest activity (15) and the up-regulation of this gene is in keeping be up-regulated, whereas average ratios of V0.5 were considered with our previous finding that antiangiogenic antithrombin to be down-regulated. We found a total of 128 genes of which inhibits the G1-to-S phase cell cycle transition in bFGF-stimulated the expression was altered based on this criterion. For most of HUVECs (10). The APC protein and tissue inhibitor of metal- these genes (f85%), native antithrombin treatment of cells loproteinase (TIMP) family proteins (TIMP-1, TIMP-2, and insignificantly altered expression relative to untreated control TIMP-3) are known inhibitors of tumor metastasis (16, 17) and cells (<1.5-fold), indicating that the majority of affected genes were caspase-3 is an important mediator of cell apoptosis (18). The only responsive to the antiangiogenically active form of anti- majority of genes with altered expression were down-regulated, thrombin and not to the inactive form. The majority of the genes with 93 of the total 128 affected genes showing a decreased affected by cleaved antithrombin are represented by five groups of expression of 2- to 4-fold in the cleaved antithrombin–treated gene clusters of which the activities are clearly implicated in cells versus the native antithrombin–treated ones. Most of those angiogenesis control (Fig. 1). The expression level of each selected genes (>80%) are in gene clusters of which the functions are gene relative to a universal mRNA control is indicated for the involved in cell adhesion, communication, migration, mitogenic three treatments by a gradient of color from red (low expression) signaling, and cell cycle control. Among those down-regulated to green (high expression). Interestingly, most of the up-regulated genes, the group of genes encoding cell-surface and matrix genes are known to play a negative role in the regulation of proteoglycans and mitogenic signaling proteins particularly stand

Figure 1. Differential expression of five groups of angiogenesis-related genes in cleaved and native antithrombin–treated HUVECs. Five groups of angiogenesis-related genes, of which the expression in cleaved antithrombin–treated HUVECs was minimally 2-fold altered from the expression in native antithrombin–treated cells in at least two of three replicate microarray chips, are tabulated from Supplementary Table S2. The expression levels of each gene in untreated, native antithrombin–, and cleaved antithrombin– treated cells relative to a universal mRNA control are depicted for each gene by the intensity of color ranging from red (lowexpression) to green (high expression) as indicated by the color key.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Research out as they represent primarily proangiogenic genes. The former treated versus native antithrombin–treated HUVECs showed a group include the HSPG family members, perlecan (HSPG2; refs. parallel alteration in expression level, we conducted Western blot 19–21), syndecan-1 (22), and syndecan-3 (23), as well as other analyses of the protein products of the affected HUVEC genes in proteoglycans such as biglycan and proteoglycan 4. The latter group the microarray and Northern blot analyses. The protein levels of of mitogenic signaling molecules include early growth response p21 WAF1/Cip1, TIMP-1, caspase-3, vitronectin, and apolipoprotein factor-1 (EGR1; ref. 24), immediate early response protein-3 (IER3/ A-1 were significantly elevated whereas the levels of STAT-3, IEX-1; ref. 25), fibroblast growth factor-20 (26), signal transducer syndecan-1, EGR1, biglycan, perlecan, and MAP kinase 3 were all and activator of transcription (STAT) family proteins 2, 3, and 6 (27), significantly decreased in cleaved antithrombin–treated cells and MAP kinase 3 (28). Supplementary Table S2 identifies the 128 relative to native antithrombin– or control-treated cells, in genes of which the expression was found to be altered by treatment agreement with the microarray and Northern blot analyses of cultured HUVECs with antiangiogenic antithrombin compared (Fig. 3). However, the protein levels for TIMP-3, APC, and with native antithrombin or control treatments of cells based on syndecan-3 were not detectably changed among cells undergoing our selection criterion. Average relative expression changes <2-fold cleaved antithrombin versus native antithrombin or control for some genes in Fig. 1 and Supplementary Table S2 reflect the fact treatments. As an internal control, h-actin protein levels remained that the differential expression in one of the three chips did not constant over the three treatment conditions. The final protein meet the 2-fold criterion. products of TIMP-3, APC, and syndecan-3 may thus be under Validation of microarray results by Northern blot and additional levels of regulatory control because the mRNA levels Western blot analyses. For an independent confirmation of the of these genes clearly differed in cleaved antithrombin–treated microarray results, we did slot Northern blot analyses of the RNA versus native antithrombin–treated cells. isolated from cultured HUVECs untreated or treated with native Validation of microarray results by quantitative real-time and cleaved antithrombins as in the microarray analyses. This was RT-PCR. Because of potential variations in the dynamic expression done using DIG-labeled cDNA probes specifically designed for patterns of endothelial cell genes affected by antiangiogenic certain selected genes of which the expression was differentially antithrombin, we wished to verify whether the 24-hour treatment regulated by cleaved and native antithrombins and of which the of HUVECs with antithrombin was appropriate to reveal the functions are clearly involved in the regulation of angiogenesis maximal changes in gene expression. Eleven genes with established (Fig. 2). The mRNA levels of eight genes found to be up-regulated roles in the regulation of angiogenesis were therefore chosen for by microarray analysis of HUVECs treated with cleaved versus real-time quantitative RT-PCR analysis. Gene-specific primers native antithrombin after 24 hours were also significantly elevated (Supplementary Table S1) were used to measure the mRNA levels by slot Northern blot analysis of these genes. Moreover, the mRNA of these genes at varying times of treatment of HUVECs with native levels of nine genes shown by microarray analysis to be down- or cleaved antithrombins (0.5, 2, 6, 12, 24, and 48 hours). mRNA regulated in cleaved antithrombin–treated versus native anti- expression levels over this time course were determined relative to thrombin–treated cells were also depressed in the slot Northern GAPDH and h-actin controls. The results clearly show substantial blots. To determine whether the protein products of the genes differential alterations of the mRNA levels of the 11 genes in a observed to be differentially expressed in cleaved antithrombin– time-dependent manner for cleaved antithrombin–treated versus

Figure 2. Slot Northern blot analysis of differentially expressed genes in cleaved antithrombin–treated versus native antithrombin–treated HUVECs. Total RNA from HUVECs after 24-hour treatment with buffer (CT), native antithrombin (ATN ), or cleaved antithrombin (ATC)was subjected to slot Northern-blot analysis using individual labeled probes specific for the indicated gene sequences. GAPDH expression was used as an internal loading control.

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Figure 3. Western blot analysis of differentially expressed genes in cleaved antithrombin–treated versus native antithrombin–treated HUVECs. Fifty micrograms of whole lysate proteins from treated or control HUVECs were subjected to 4% to 12% gradient SDS-PAGE, transferred to Immobilon P membranes, and individual proteins were detected using specific antibodies. h-Actin protein levels were determined as an internal loading control.

native antithrombin–treated HUVECs, with the maximal changes gene transcription relative to controls but induced the expression of gene expression occurring for most genes at 24 hours (Fig. 4). of the proapoptotic genes, Max and Bax, by 1.5- to 2.5-fold. Exceptions were p21WAF1/Cip1 of which the maximal induction by Effect of bFGF on the gene expression profile of HUVECs cleaved antithrombin occurred relatively early, f6 hours, following treated with cleaved antithrombin. Because previous studies treatment and vitronectin, which was induced relatively late, its showed the antiangiogenic effects of conformationally altered expression peaking at 48 hours. Interestingly, syndecan-1 gene forms of antithrombin mostly in growth factor (VEGF/bFGF)– expression was dramatically induced in both cleaved antithrom- stimulated endothelial cells (5–7, 10), we determined the effects of bin–treated and native antithrombin–treated HUVECs over the bFGF on the differential expression of genes in HUVECs treated initial 12-hour period and became down-regulated in cleaved with cleaved antithrombin versus native antithrombin or buffer antithrombin–treated versus native antithrombin–treated cells using the microarray technique. Based on our 2.0-fold change only at the 24- and 48-hour time points. Overall, the kinetic selection criterion, we found that cleaved antithrombin resulted in analysis of gene expression for the 11 selected genes confirmed the an altered expression of 136 genes relative to native antithrombin reliability of the HUVEC gene expression patterns revealed by treatment of cells stimulated with bFGF (data not shown). microarray analysis after 24-hour treatment of cells with anti- However, only 15 of these genes were identical to those thrombin. differentially expressed in unstimulated HUVECs (Table 1). bFGF Cleaved antithrombin–induced apoptosis in HUVECs. Previ- enhanced or produced a similar differential expression of 10 genes ous studies have shown that antiangiogenic forms of antithrombin in cleaved antithrombin–treated versus native antithrombin– induce apoptotic cell death in cultured bovine capillary endothelial treated cells including the angiogenesis-related genes, APC, cells (6) and in tumors implanted in mice (9). Interestingly, our caspase-3, CDCA3, EGR1, HSPG2, and IGFBP1. There were two microarray, slot Northern blot, Western blot, and quantitative real- genes of which the altered expression in cleaved antithrombin– time RT-PCR analyses all showed that caspase-3 gene expression treated versus native antithrombin–treated cells was attenuated in was induced >2-fold in HUVECs treated with cleaved antithrombin the presence of bFGF. Interestingly, bFGF reversed the effects of versus native antithrombin or control for 24 hours. To further cleaved antithrombin on the expression of three genes which were confirm the apoptotic effects of cleaved antithrombin on mostly not angiogenesis related. These results indicate that bFGF endothelial cells, we quantitated apoptosis in antithrombin-treated modulates the effects of antiangiogenic antithrombin on endo- and untreated HUVECs. HUVECs treated with cleaved antithrom- thelial cell gene expression and results in more pronounced effects bin for 24 hours showed >2-fold increase in apoptotic death on several angiogenesis-related genes. relative to control-treated cells (Fig. 5A and B). Moreover, cleavage of poly(ADP-ribose) polymerase, a landmark of cell apoptosis (29), was elevated in cleaved antithrombin–treated cells relative to Discussion controls (Fig. 5C). Because other apoptosis-related genes may have The present study was designed to identify target genes escaped detection in our microarray analysis, we determined the affected by treatment of primary HUVECs with antiangiogenic mRNA levels of four representative genes including those in the Bcl antithrombin and thereby to reveal how endothelial cell gene family (i.e., p53, Bcl-2, Max, and Bax; ref. 30) by quantitative real- expression is reprogrammed to orchestrate the antiangiogenic time RT-PCR analysis. The results (Fig. 5D) show that cleaved response. Our previous report showed that antiangiogenic antithrombin produced no significant alterations in Bcl-2 and p53 cleaved and latent forms of antithrombin significantly suppressed www.aacrjournals.org 5051 Cancer Res 2006; 66: (10). May 15, 2006

Figure 4. Kinetics of expression of selected genes in antithrombin-treated and untreated HUVECs. Real-time quantitative RT-PCR analysis was done on total RNA samples from HUVECs treated with 30 Ag/mL cleaved antithrombin, native antithrombin, or untreated control for 0.5, 2, 6, 12, 24, and 48 hours. Columns, average expression levels in quadruplicate samples from cleaved antithrombin–treated cells (black columns) and from native antithrombin–treated cells (white columns), normalized to the expression levels from control untreated cells (gray columns); bars, SD. the expression of the proangiogenic HSPG, perlecan, in HUVECs endothelial cells are exposed to f100 Ag/mL levels of the independent of whether the cells were stimulated with growth protein in circulating blood under normal physiologic conditions factor (10). This clearly showed that the effects of antiangiogenic (4). For those genes of which the expression was significantly antithrombin were not simply mediated by blocking growth altered by cleaved antithrombin treatment of HUVECs, native factor effects on endothelial cells but involved changes in antithrombin treatment typically showed marginal changes from endothelial cell gene expression presumably requiring signaling untreated cells, consistent with the effects being specific for the through a receptor. Our current study more firmly establishes conformationally altered form of the serpin. This sets antithrom- that the antiangiogenic cleaved form of antithrombin significant- bin apart from other antiangiogenic serpins of which the ly alters the expression not only of the perlecan gene but also of antiangiogenic activity is not conformation dependent. Selected numerous other genes in endothelial cells, strongly implicating genes of which the expression was altered in the microarray an endothelial cell ligand-receptor signaling mechanism in experiments by cleaved antithrombin treatment and which have mediating these global changes in gene expression. This idea is established roles in angiogenesis were confirmed to have altered supported by our finding that the majority of changes in gene expression by Northern blot and real-time RT-PCR analyses and, expression induced by cleaved antithrombin involved the down- in most cases, also altered protein levels by Western blot regulation of proangiogenic genes and the up-regulation of analyses. Cleaved antithrombin thus seems to induce an antiangiogenic genes, in keeping with the antiangiogenic activity antiangiogenic signaling network in endothelial cells resembling of the protein. Our selection criterion for affected genes required in its overall effect that induced by the angiogenesis inhibitor, that minimally 2-fold expression changes in at least two endostatin (31), although the specific genes affected differ for the microarray chips be observed in cleaved antithrombin–treated two proteins. Although the effects of the antiangiogenic latent cells relative to cells treated with native antithrombin. Native form of antithrombin on endothelial cell gene expression were antithrombin was considered the best reference state because not tested in the present study, it should be noted that cleaved this form of the protein lacks antiangiogenic activity and and latent antithrombin forms have previously been shown to

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Antiangiogenic Antithrombin Gene Expression Profiling display indistinguishable antiangiogenic activities in proliferation, effect by blocking endothelial cell integrins required for growth. migration, capillary-like tube formation, bFGF signaling, and Integrin blockade underlies the mechanism of antiangiogenic perlecan gene expression assays of angiogenesis (6, 7, 10). action of several other natural angiogenesis inhibitors (37). Our results show that several genes clustered in the cell matrix Together, the alterations in the expression of endothelial cell protein group, including perlecan (HSPG2), syndecan-1, syndecan- matrix molecules induced by antiangiogenic antithrombin may 3, biglycan, proteoglycan-4, and a5-laminin, were all down- contribute to the reported attenuating effects of latent antithrom- regulated by >2-fold. It has been clear that most of the molecular bin on focal adhesion kinase activation (6). events associated with tumor growth, neovascularization, and Antiangiogenic forms of antithrombin have been shown to metastasis are influenced by interactions between cancer cells and induce endothelial cell apoptotic death in vitro (6) and in vivo (9), their extracellular matrix components (32). HSPGs such as consistent with our current microarray data that the expression of perlecan, syndecan-1, and syndecan-3 can act both as reservoirs caspase-3 and several other proapoptotic molecules is significantly for growth factors and as coreceptors for ligand binding and induced in cleaved antithrombin–treated cells. Furthermore, our subsequent intracellular signaling (33). Therefore, reductions of quantitative real-time RT-PCR analysis also indicated that the these HSPG molecules on endothelial cells have been considered ratio of Bcl-2/Bax in endothelial cells treated with cleaved as an efficient approach to control cell growth, invasion, antithrombin was decreased, which is a well-known indicator of angiogenesis, and tumor progression (34). Intriguingly, the matrix apoptosis (30). Previous studies have shown that numerous HSPG, agrin, was found to be up-regulated by antiangiogenic angiogenesis inhibitors, including several serpins, exert their antithrombin, suggesting that this HSPG may not be proangio- antiagiogenic effects partly by inducing endothelial cell apoptosis genic. The a5-laminins are prominent basement membrane (38–41). components which promote bFGF- and VEGF-induced endothelial We previously showed that antiangiogenic forms of antithrom- a h cell growth via ligation of v 3 integrins (35). An indirect bin inhibit growth factor–stimulated cell proliferation by inhibitory effect on the expression of other endothelial cell arresting the cell cycle at the G1-S phase (10). The present study adhesion molecules is also expected based on our observation shows that the antiangiogenic cleaved form of antithrombin acts that the InBq inhibitor of NF-nB, which controls NF-nB-dependent directly to inhibit cell growth by down-regulating cell cycle expression of adhesion molecules, is up-regulated by antiangio- regulatory proteins which promote growth and by up-regulating genic antithrombin (Supplementary Table S2; ref. 36). Interestingly, proteins which inhibit growth (42). The antiangiogenic activity of our microarray results show that certain matrix molecules such as endostatin is similarly mediated by altering the expression of cell vitronectin were up-regulated >2-fold in cleaved antithrombin– cycle control proteins in endothelial cells to favor a quiescent treated versus native antithrombin–treated cells. A previous report state (31). has shown that vitronectin is essential for cleaved antithrombin to The cytokine-induced oligomerization of receptor subunits express its antiangiogenic function in vivo (14). It has been and engagement of an intracellular signaling machinery is a suggested that vitronectin forms a complex with antiangiogenic common feature by which cytokines such as bFGF and VEGF antithrombin either directly or indirectly through the binding of induce mitogenic effects in target cells (43). Cytokine growth both proteins to heparin, which then exerts an antiangiogenic factors may signal through several parallel MAP kinase pathways

Figure 5. Antiangiogenic antithrombin induces endothelial cell apoptosis. A, primary HUVECs were cultured in serum-reduced medium in the presence or absence of 30 Ag/mL of native or cleaved antithrombin for 24 hours. The apoptotic bodies of antithrombin-treated HUVECs were detected by fluorescence microscopy after staining with DAPI. B, columns, mean percentage of apoptotic cells in HUVECs subjected to control, native antithrombin, or cleaved antithrombin treatments, quantified by counting at least four fields of cells; bars, SD. *, P < 0.01. C, cleavage of poly(ADP-ribose) polymerase (PARP) in HUVECs treated for 24 hours as in (A and B) assessed by Western blot analysis. D, real-time quantitative RT-PCR analysis of mRNA expression of apoptosis-related genes in HUVECs treated with buffer (blank columns), native antithrombin (gray columns), and cleaved antithrombin (black columns). Columns, mean of three assays; bars, SD. *, P < 0.05.

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Table 1. Effects of bFGF on the changes in HUVEC gene The matrix metalloproteinases (MMP) play an important role in expression induced by cleaved antithrombin tumor invasion by functioning to degrade the macromolecules of the extracellular matrix. Abundant evidence indicates that Gene symbol Cluster ID Fold change controlling the activity of MMPs by maintaining their zymogen state (pro-MMPs) or inactivating the active MMPs is an effective ÀbFGF +bFGF approach to control angiogenesis and tumor metastasis (53). The naturally occurring inhibitors, TIMPs, are important regulators of Enhanced or unchanged the activities of MMPs in normal and disease processes (17). Thus APC Hs.158932 2.3 3.1 far, four distinct TIMPs (TIMP-1, TIMP-2, TIMP-3, and TIMP-4) APOA1 Hs.93194 2.0 5.7 have been isolated, cloned, and characterized in several species. CASP3 Hs.141125 2.5 3.3 Interestingly, cleaved antithrombin was found to induce the CDCA3 Hs.524216 0.49 0.25 expression of three TIMPs >2-fold in HUVECs and the induction CYP111B1 Hs.184927 0.24 0.24 of TIMP-1 and TIMP-3 was confirmed at the mRNA level and, in EGR1 Hs.326035 0.41 0.23 GPR143 Hs.74124 0.48 0.29 the case of TIMP-1, also at the protein level. Because the activities HSPG2 Hs.555874 0.44 0.24 of MMPs are strictly regulated by TIMPs, these results may at least in vivo IGFBP1 Hs.401316 0.59 0.22 partly explain the previous observations that antiangiogenic NYD-SP18 Hs.131098 0.43 0.20 forms of antithrombin induce significant tumor regression in mice Attenuated (5, 6, 8, 9). The control of tumor metastasis in certain cancers by FCER1G Hs.433300 0.30 0.46 inducing endogenous TIMP expression or delivering exogenous NIN Hs.310429 0.45 0.50 TIMPs has become a promising cancer therapy approach (54). Reversed Our data thus support the potential efficacy of antiangiogenic HIPK2 Hs.397465 0.25 6.62 antithrombin as an antitumor drug. IGLL1 Hs.348935 0.28 4.18 Antiangiogenic forms of antithrombin significantly inhibit PCSK2 Hs.315186 2.17 0.15 growth factor–stimulated proangiogenic activities including cell proliferation, migration, capillary tube formation, and growth factor–induced signaling (5–7, 10). Whereas a comparable number of genes were found to be differentially expressed in bFGF- to induce cytokine-specific gene transcription patterns or through stimulated HUVECs treated with cleaved antithrombin versus receptor-associated Janus kinases, which phosphorylate STAT native antithrombin, only f10% of these genes were identical to family transcription factors, to induce gene transcription (44, 45). those differentially expressed in unstimulated HUVECs. Several of Compared with normal cells and tissues, constitutively activated the angiogenesis-related genes, including APC, caspase-3, perlecan, MAP kinases and/or STATs have been detected in a wide variety and EGR-1, showed an even greater differential expression in of human cancer cell lines and primary tumors. It is clear that cleaved antithrombin–treated versus native antithrombin–treated MAP kinases and STATs, particularly STAT-3, play an important cells stimulated with bFGF than cells not stimulated. The role in growth factor–mediated angiogenesis (27, 43, 46). Our remaining f90% of genes with significantly altered expression microarray results, partially confirmed by real-time quantitative in unstimulated, but not stimulated, cells may have fallen below RT-PCR, Northern-blot, and/or Western blot analysis, show that the threshold of our selection criterion for altered expression 26 mitogenesis-related signaling molecule genes were significantly because of the up-regulation of a wide range of endothelial cell down-regulated in endothelial cells following cleaved antithrom- genes in control bFGF-stimulated cells. The effects of endostatin bin treatment. Among these down-regulated genes, the transcrip- on endothelial cell gene expression also were determined in the tional regulators EGR1 and IER3/IEX-1 are downstream targets of absence of growth factor stimulation presumably to more clearly extracellular signal–regulated protein kinase 1/2, the protein reveal its antiangiogenic signaling effects (31). That the differential kinase that is activated by mitogens like bFGF/VEGF (25, 47). The effects of cleaved antithrombin versus native antithrombin on biological function of EGR1 has been closely connected with the gene expression observed in unstimulated cells are likely to be development of human cancers because overexpression of EGR1 important also in stimulated cells is suggested by our recent in a majority of human prostate cancers has been observed (48). finding that antiangiogenic forms of antithrombin inhibit bFGF- Moreover, tumor progression in transgenic mouse models of induced proangiogenic effects by competing with bFGF for prostate cancer was reported to be significantly impaired when binding a common heparan sulfate coreceptor.5 The observed EGR1 was lacking (49). The down-regulation of EGR1 gene effects of cleaved antithrombin on gene expression in bFGF- expression in antiangiogenic antithrombin–treated cells was fully stimulated HUVECs are thus likely to reflect a balance between confirmed at the mRNA and protein levels and this down- reversing the effects of growth factor and the direct effects regulation was shown to be further enhanced by bFGF mediated through an HSPG receptor or coreceptor. stimulation of HUVECs (Table 1). Interestingly, EGR1 gene In summary, our microarray results show that the antiangiogenic expression is significantly suppressed in pigment epithelium– cleaved form of antithrombin exerts its antiangiogenic function by derived factor–treated cells (50), another potent antiangiogenic globally altering gene expression in cultured HUVECs, mainly by serpin. Two down-regulated mitogenic transcription factors of the suppressing proangiogenic genes, including cell-surface HSPGs and ets family, SPDEF and ELK1, are downstream targets of MAP MAP kinase and STAT signaling molecules, and by inducing kinase pathways and found to be up-regulated in cancer (51, 52). Together, these results suggest the importance of down-regulating the MAP kinase– and STAT-dependent signaling pathways to mediate the antiangiogenic activity of antithrombin. 5 W. Zhang, R. Swanson, Y. Xiong, S.T. Olson, submitted for publication.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Antiangiogenic Antithrombin Gene Expression Profiling antiangiogenic genes, including tumor suppressors, proapoptotic, Acknowledgments and cell cycle control proteins. The genetic reprogramming of Received 12/20/2005; revised 3/1/2006; accepted 3/13/2006. endothelial cells by antiangiogenic antithrombin revealed in this Grant support: NIH grant HL-39888 (S.T. Olson). study suggests that certain target genes are involved in mediating The costs of publication of this article were defrayed in part by the payment of page the antiangiogenic response. Further studies will be required to charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. decipher how the protein products of these genes accomplish this We thank Dr. Peter Gettins (University of Illinois at Chicago) for helpful comments function. on the manuscript.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Antiangiogenic Antithrombin Induces Global Changes in the Gene Expression Profile of Endothelial Cells

Weiqing Zhang, Yung-Jen Chuang, Tianquan Jin, et al.

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