This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Caunt, M. Articles by Karpatkin, S. Search for Related Content PubMed PubMed Citation Articles by Caunt, M. Articles by Karpatkin, S.

Growth-Regulated Oncogene Is Pivotal in Thrombin-Induced Angiogenesis

Departments of ¹ Medicine, ² Radiation Oncology and Cell Biology, and ³ Pathology, New York University School of Medicine, New York, New York

Requests for reprints: Simon Karpatkin, Department of Medicine, New York University School of Medicine, 550 First Avenue, New York, NY 10016. Phone: 212-263-5609; Fax: 212-263-0695; E-mail: simon.karpatkin{at}med.nyu.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The mechanism of thrombin-induced angiogenesis is poorly understood. Using a gene chip array to investigate the pro-malignant phenotype of thrombin-stimulated cells, we observed that thrombin markedly up-regulates growth-regulated oncogene- (GRO-) in several tumor cell lines as well as endothelial cells by mRNA and protein analysis. Thrombin enhanced the secretion of GRO- from tumor cells 25- to 64-fold. GRO- is a CXC chemokine with tumor-associated angiogenic as well as oncogenic activation following ligation of its CXCR2 receptor. GRO- enhanced angiogenesis in the chick chorioallantoic membrane assay 2.2-fold, providing direct evidence for GRO- as an angiogenic growth factor. Anti-GRO- antibody completely inhibited the 2.7-fold thrombin-induced up-regulation of angiogenesis, as well as the 1.5-fold thrombin-induced up-regulation of both endothelial cell cord formation in Matrigel and growth in vitro. Thrombin as well as its PAR-1 receptor activation peptide [thrombin receptor activation peptide (TRAP)] as well as GRO- all markedly increased vascular regulatory proteins and growth factors: matrix metalloproteinase (MMP)-1, MMP-2, vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), CD31, and receptors KDR and CXCR2 in human umbilical vein endothelial cells. All of the thrombin/TRAP gene up-regulations were completely inhibited by anti-GRO- antibody and unaffected by irrelevant antibody. Similar inhibition of gene up-regulation as well as thrombin-induced chemotaxis was noted with small interfering RNA (shRNA) GRO- KD 4T1 breast tumor and B16F10 melanoma cells. In vivo tumor growth studies in wild-type mice with shRNA GRO- KD cells revealed 2- to 4-fold impaired tumor growth, metastasis, and angiogenesis, which was not affected by endogenous thrombin. Thus, thrombin-induced angiogenesis requires the up-regulation of GRO-. Thrombin up-regulation of GRO- in tumor cells as well as endothelial cells contributes to tumor angiogenesis. (Cancer Res 2006; 66(8): 4125-32)

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The promalignant role of thrombin in tumor adhesion (1–4), growth (5), metastasis (1, 2, 6–9), and angiogenesis (10–15) is well recognized. However, the mechanism of thrombin-induced angiogenesis is not clear. Vascular regulatory proteins and growth factors, particularly matrix metalloproteinases (MMP-1, MMP-2, and MMP-9), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), and Ang-2, are required for the regulation of blood vessel formation.

Thrombin-induced angiogenesis in a chick chorioallantoic membrane (CAM) assay is associated with up-regulation of the major VEGF (15) as well as Ang-2 (15). Thrombin also up-regulates VEGF (10, 16) and the major VEGF receptor KDR in endothelial cells (14) and induces the secretion of VEGF (17, 18) and Ang-1 (19) from platelets. Thrombin up-regulates Ang-2, MMP-1, and MMP-2 in endothelial cells (11, 20). However, the cellular mechanisms responsible for thrombin-induced up-regulation of these genes have not been established.

The chemokine growth-regulated oncogene- (GRO-) has been implicated in blood vessel formation. GRO- is a CXC chemokine with oncogenic activity. GRO--induced metastasis is associated with increased angiogenesis (21–23). GRO- binds to a seven-transmembrane receptor, CXCR2 on endothelial cells and neutrophils, and promotes their chemotaxis (24). It is required for maintenance of wound repair and is necessary for neutrophil recruitment (25). GRO- enhances growth, chemotaxis, and metastasis of several tumor cell lines (22, 23, 26–28).

Using an Affymetrix gene chip array to investigate the promalignant phenotype of thrombin-stimulated cells, we observed that thrombin up-regulates GRO- 168-fold in an undifferentiated mouse tumor cell line (UMCL) and confirmed by reverse transcription-PCR (RT-PCR). Thrombin-induced up-regulation of GRO- mRNA and protein was also noted in several other tumor cell lines, as well as human umbilical vein endothelial cells (HUVEC).

In this article, we show that (a) thrombin induces the up-regulation and secretion of GRO- from tumor cells as well as HUVECs; (b) GRO-, like thrombin, enhances neoangiogenesis, endothelial cord formation, and endothelial cell growth; (c) GRO-, like thrombin, up-regulates an identical series of five proangiogenic genes; (d) anti-GRO- antibody or small interfering RNA (siRNA) GRO- knockdown (KD) inhibits the up-regulation of these genes as well as thrombin-induced angiogenesis, endothelial cord formation, chemotaxis, and cell growth; (e) thrombin-enhanced tumor growth, angiogenesis, metastasis, and up-regulation of VEGF and Ang-2 is inhibited in wild-type mice using GRO- KD 4T1 breast carcinoma cells. These data indicate a pivotal role for GRO- in thrombin-induced angiogenesis.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents. Thrombin and thrombin receptor activation peptide SFLLRNPNDKYEPF (TRAP) were purchased from Sigma Chemical Co. (St. Louis, MO). The irrelevant control peptide, CAPESIEFPVSEARVLED, was synthesized by Quality Control Biochemicals (Hopkinton, MA). Hirudin (Refludan) was obtained from Hoechst Marion Rousel (Kansas City, MO). Antibodies to Ang-2, CD31, CD105, von Willebrand factor (VWF), KDR, VEGF, ß-actin, and tubulin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to MMP-1 and MMP-2 were purchased from Chemicon (Temecula, CA). Human recombinant GRO-, basic fibroblast growth factor (bFGF), and anti-GRO- were purchased from Biosource International (Camarillo, CA). Control IgG was purchased from Pierce (Rockford, IL). Growth factor–reduced Matrigel was purchased from Becton Dickinson (Lincoln Park, NJ). The GRO- ELISA was done with a kit from R&D Systems (Minneapolis, MN).

Cell lines and culture conditions. Human prostate cell line PC3 and breast carcinoma cell line MCF-7 and murine B16F10 melanoma and breast 4T1 cells were purchased from American Type Culture Collection (Manassas, VA) and maintained in DMEM (Sigma Chemical) supplemented with 10% fetal bovine serum (FBS; Life Technologies), 2 mmol/L L-glutamine, and penicillin-streptomycin. HUVECs were obtained from Cambrex Bioscience (Walkersville, MD) and were maintained in EBM-2 (Cambrex Bioscience) supplemented with 2% FBS and growth factors (singlequots) according to the instructions of the manufacturer. Human brain microvascular endothelial cells (HBMEC) were provided by Dr. Jorge Ghiso (New York University Medical Center, New York, NY). All cells were grown at 37°C in 5% CO₂. Tumor cells were starved for 17 hours and HUVECs for 4 hours in the absence of FBS before incubation with agonists.

Chick CAM angiogenesis assay. Angiogenesis assays were done as previously described (15). Briefly, 10-day-old chick embryos were prepared by separating the CAM from the shell membrane. Filter discs were placed on the CAM and the test compound or PBS was added to the disc at 0, 24, and 48 hours for thrombin and GRO- experiments. For bFGF experiments, the filter disc was soaked in bFGF in RPMI for 30 minutes before addition to the CAM. Embryos were sacrificed at 72 hours and the CAM was removed for analysis. Angiogenesis was quantified by counting the number of branching blood vessels within the confined area of the filter disc by stereomicroscopy. Each experiment was completed thrice, with 8 to 12 embryos per condition. Results are mean ± SE of the angiogenic index.

Endothelial cell cord formation. Matrigel (100 µL) was added to a 96-well plate and left to polymerize for 1 hour at 37°C. HUVECs were trypsinized and resuspended in EBM-2, 2% FBS with or without thrombin, hirudin, or anti-GRO- antibody. One hundred microliters were added to the top of the Matrigel and incubated for 24 hours at 37°C. Branch points were analyzed with an inverted microscope by phase microscopy and counted in four random fields per well. All measurements were made in triplicate.

Endothelial cell culture in vitro. HUVECs were grown to near confluence, starved for 4 hours in the absence of FBS, and then incubated for 48 hours in the presence of GRO-, thrombin, or GRO- antibody or irrelevant IgG (2 µg/mL). Growth was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (A11) with a Cell Proliferation kit supplied by Roche Diagnostic Corp. (Indianapolis, IN).

Chemotaxis assay. Transwell plates (obtained from Costar 3422, Corning, Inc., Corning, NY) were used to measure tumor chemotaxis. Cells were grown to 75% confluence in 10% bovine serum albumin (BSA)-DMEM culture medium for 24 hours, followed by 0.1% BSA-DMEM for an additional 24 hours. Cells were then trypsinized, washed with PBS, resuspended in 0.1% BSA-DMEM, and 200 µL (5 x 10⁴ cells) were added to the upper chamber. The lower chamber contained 0.1 unit/mL thrombin in 600 µL of 0.1% BSA-DMEM. Plates were incubated at 37°C, 5% CO₂, for 3 hours. Inserts were removed, washed with PBS, and then stained with crystal violet for 10 minutes. Cells and solutions had been removed from the inside of the insert. Excess stain was removed from the bottom of the insert by swabbing with a cotton-tipped applicator and then allowed to dry. Destaining was performed in 10% acetic acid for 10 minutes. The solution was then transferred to a 96-well plate and absorbance was read at 595 nm.

RNA analysis. RT-PCR for human GRO- (J03561) was done with the following primers: forward 5'-TTCACCCCAAGAACATCCAAAG-3' and reverse 5'-CAAACACATTAGGCACAATCCAGG-3', which amplifies a 291 bp fragment of GRO-. RT-PCR for mouse GRO- (J04596) used the following primers: forward 5'-GCACCCAAACCGAAGTCATAGC-3' and reverse 5'-TTGTCAGAAGCCAGCGTTCAG-3', which amplifies a 175 bp fragment.

RT-PCR for CD105 used the following primers:

Forward: 5'-CTTCCAAGGACAGCCAAGAG-3'.

Reverse: 5'-GTGGTTGCCATTCAAGTGTG-3'.

RT-PCR for VWF used the following primers:

Forward: 5'-GCTTCTTACGCCCATCTCTG-3'.

Reverse: 5'-CAGCTGCCTTCCAGAAAAAC-3'.

Knock down of GRO- in 4T1 cells by shRNA. GRO- shRNA was introduced into the shRNA-RetroQ viral vector (BD Biosciences, Clontech, San Diego, CA) at the BamH1 and EcoR1 ligation sites according to the directions of the manufacturer. shRNA oligonucleotides were derived from the murine GRO- sequence (NIH Genbank accession no. J04596) and synthesized following derivation from the computer program supplied by BD Biosciences. Successful inhibitory siRNAs include the following:

G2 strand sequence:

5'-GATCCGCAGAGAGATAGAGTTTAGTAttcaagagaTACTAAACTCTATCTCTCTGCTTTTTTG-3' plus complementary strand,

5'-AATTCAAAAAAGCAGAGAGATAGAGTTTAGTAtctcttgaaTACTAAACTCTATCTCTCTGCG-3'.

G8 strand sequence:

5'-GATCCGCTTCTGACAACACTATACAAttcaagagaTTGTATAGTGTTGTCAGAAGCTTTTTTG-3' plus complementary strand,

5'-AATTCAAAAAAGCTTCTGACAACACTATACAAtctcttgaaTTGTATAGTGTTGTCAGAAGCG-3'.

hG2 strand sequence:

5'-GATCCGCATCGCTTAGGAGAAGTCTTttcaagagaAAGACTTCTCCTAAGCGATGCTTTTTTG-3' plus complementary strand,

5'-AATTCAAAAAAGCATCGCTTAGGAGAAGTCTTtctcttgaaAAGACTTCTCCTAAGCGATGCG-3'.

A scrambled oligonucleotide was used as a control:

5'-GATCCAGCTCGCGTTTATCAGAGACTttcaagagaAGTCTCTGATAAACGCGAGCTTTTTTT-3' plus complementary strand,

5'-AATTCAAAAAAAGCTCGCGTTTATCAGAGACTtctcttgaaAGTCTCTGATAAACGCGAGCTG-3'.

The two paired oligonucleotides were annealed to form double strands. Verification of the inserted sequence was obtained by automated DNA sequencing from the Core Laboratory at New York University Medical Center. The plasmids were packaged into Phoenix Ampho cells (BD Biosciences) by standard calcium phosphate transfection. Virus supernatants were collected at 48 hours posttransfection, centrifuged to remove nonadherent cells and cellular debris, and frozen in small aliquots at –80°C.

4T1 cells were seeded at 20,000 per well in 24-well plates. The following day, the culture medium was aspirated and replaced with retroviral supernatant diluted 1:2 DMEM in to a final volume of 1 mL in growth medium (DMEM) plus 10% FBS and penicillin-streptomycin. Polybrene was added to a final concentration of 4 µg/mL. Twenty-four hours after infection, the cells were collected by typsinization and reseeded in six-well dishes in selective medium + 1 µg/mL puromycin. Single-cell colonies were picked and reseeded with selective medium.

Traditional RT-PCR and real-time RT-PCR were done to validate KD of GRO- mRNA as well as to measure the effect of thrombin on GRO- KD 4T1 cells. iCycle iQ real-time PCR (iScript one-step RT-PCR with SYBR green) was purchased from Bio-Rad (Hercules, CA) and done according to the instructions of the manufacturer.

Immunoprecipitation and Western blotting. HUVECs were starved for 4 hours, then medium was removed and then replaced with added 5% BSA plus agonist studied. Tumor cells were starved for 17 hours and agonist was added for an additional 24 hours. Total cell extracts were prepared after removal of medium and lysing of cells in lysis buffer containing 1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L EGTA, 2.5 mmol/L sodium PPi, 1 mm ß-glycerol phosphate, 50 mmol/L Tris-HCL (pH 7.5), 10% glycerol, 1 mmol/L Na₃VO₄, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 µg/mL leupeptin. After centrifugation, the supernatants (1 mg/mL) were incubated with 3 µL antibody (200 µg/mL) overnight at 4°C, followed by the addition of 30 µL protein A/G beads (0.5 mL/mL; Santa Cruz Biotechnology), and further incubated at 4°C for 2 hours followed by centrifugation. The immune complexes (beads) were washed thrice with lysis buffer and then suspended in 50 µL SDS-PAGE loading buffer. The washed, suspended beads were boiled at 95°C for 5 minutes, chilled at 4°C, and centrifuged. Thirty microliters of supernatant were then run on 10% SDS-PAGE under reducing conditions and transferred to a nitrocellulose membrane. Membranes were incubated with the appropriate antibody (1 µg/mL) for 1 hour at room temperature, washed, and incubated with horseradish peroxidase (HRP)–conjugated secondary antibody for another hour and developed by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ).

Immunoassay for GRO-. Cells were serum starved for 4 hours before treatment with thrombin for 4 hours and conditioned medium was collected. ELISA for human GRO- was done using Quantikine human GRO- Immunoassay (R&D Systems).

In vivo studies in mice. 4T1 cells were used because they induce spontaneous metastasis in syngeneic mice without the need to sacrifice animals because of enhanced tumor burden. 4T1-green fluorescent protein (GFP)–labeled cells were prepared as described (8). These cells were infected with shRNA GRO- retrovirus or empty vector. GRO- KD-GFP cells (1 x 10⁵) or empty vector–GFP cells were injected s.c. into BALB/c mice as described previously (8). Both groups were also treated with and without hirudin, 10 mg/kg i.p, daily for 10 days followed by every other day for 7 days. Day 1 injection was given 5 minutes before and 4 hours after tumor inoculation. Animals were sacrificed on day 25 and their spontaneous pulmonary metastasis was evaluated as described (8). Tumor detection in the blood was determined by flow cytometry of GFP-labeled 4T1 cells on day 24 as previously described (8). The flow cytometry was gated with standard GFP cells before enumeration of cells in the blood. Ten thousand events were accumulated for each sample. Approximately 80% of infected cells were GFP positive.

Immunohistochemical staining. Tumor nodules were fixed in 4% formalin. Paraffin-embedded tissue sections were used for H&E as well as anti-VWF immunnohistochemical staining. The stains were done on an automated autostainer (Ventana Medical Systems, Tuscon, AZ) according to the instructions of the manufacturer. Primary antibody titer was determined from a dilution curve. Optimum staining was obtained at a dilution of 1:40, which gave the lowest background and best sensitivity for antigen retrieval. Rabbit polyclonal anti-VWF IgG was followed by a biotinylated goat anti-rabbit polyclonal antibody, followed by streptavidin-HRP and H₂O₂ substrate plus 3,3'-diaminobenzidine developing chromagen, which produces a brown precipitate. The entire tissue section was examined and areas with the highest intensity of staining (i.e., hotspots; ref. 29) were identified and further examined under high-power magnification (x400) for VWF expression. The number and intensity of staining of small vessels in the hotspots were scored in a blinded fashion. An angiogenesis index was obtained by multiplying the number of vessels by the intensity of staining (scored 1-3).

Statistical analysis. This was done by Student's t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin up-regulates GRO- mRNA and protein in tumor and endothelial cells. RT-PCR, Northern blot analysis, and immunoblotting were done on thrombin-stimulated tumor cell lines and HUVECs to confirm the gene chip array data and test for a generic effect in multiple cell lines. Figure 1A shows thrombin-induced mRNA up-regulation of GRO- as measured by semiquantitative RT-PCR in UMCL, B16F10, and MCF7 cells at 24 hours. Figure 1B shows that thrombin or TRAP also induce up-regulation of GRO- protein in PC3 cells 3.4- and 3.1-fold, respectively, by immunoblot at 24 hours. Similar up-regulation of 2.0- and 1.8-fold was noted with HUVECs at 0.1 and 0.5 unit/mL, respectively. Similar up-regulation of 2.4-fold was also noted with primary endothelial cells obtained from a second source, HBMECs treated with 0.5 unit/mL thrombin. Figure 1C shows concentration-dependent, thrombin-induced up-regulation of GRO- secretion with 0.5 and 1 unit/mL thrombin by ELISA assay in MCF7 cells at 24 hours of 9- and 25-fold, respectively, as well as in PC3 cells of 36- and 64-fold, respectively. These data indicate that thrombin can induce various tumor cells to produce GRO-.

View larger version (15K): [in this window] [in a new window]   Figure 1. Up-regulation of GRO- by thrombin. A, semiquantitative RT-PCR for GRO- in murine B16F10 melanoma, UMCL, and human MCF7 breast cells. B16F10 cells were incubated with buffer 0.1, 0.5, 1, 1.5, and 2 units/mL thrombin for 24 hours. UMCL and MCF7 cells were incubated with buffer, and 0.1 and 0.5 unit/mL thrombin. Actin or GAPDH was used as loading control. Representative of two to three experiments. B, immunoblot of GRO- in PC3 cells incubated with buffer, 100 µmol/L TRAP (TRP) or 0.5 unit/mL thrombin (0.5T) for 24 hours using tubulin as loading control (Ctl). Immunoblot of HUVECs incubated with 0.1 and 0.5 unit/mL thrombin. Representative of two experiments. Immunoblot of HBMECs incubated with 0.5 unit/mL thrombin. C, ELISA assay for GRO- secretion in 1 x 106 MCF7 or PC3 cells at 24 hours using 0.5 and 1 unit/mL (1T) thrombin (n = 3).

GRO- and thrombin-induced angiogenesis and endothelial cell growth is inhibited by anti-GRO- antibody.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of GRO-, thrombin, and anti-GRO- antibody on angiogenesis in the CAM, endothelial cord formation, and endothelial cell growth assays. A, GRO- activation of angiogenesis at 72 hours and 37°C in the CAM assay (25 and 50 ng/egg, n = 19). B, inhibition of thrombin-induced angiogenesis (Thr) by anti-GRO- antibody (T+GROAb, 5 µg/egg) or irrelevant IgG (T+IgG), n = 9. C, effect of thrombin (0.25 unit/mL), thrombin plus 1 unit/mL hirudin (T+H), or hirudin (H) on thrombin-induced cord formation of endothelial cells (n = 20). Effect of GRO- (2 µg/mL), thrombin plus anti-GRO- antibody (T+Ab, 5 µg/mL), or control plus anti-GRO- antibody (C+Ab, 5 µg/mL) on thrombin-induced cord formation of endothelial cells in Matrigel at 24 hours and 37°C (n = 7). D, effect of GRO- (0.025-0.5 µg/mL; Gro) or thrombin (T, 0.5 unit/mL) on HUVEC growth in vitro at 48 hours and 37°C. Inhibition of thrombin-induced HUVEC growth in the presence of 5 µg/mL anti-GRO- antibody. Absence of inhibition in the presence of irrelevant IgG (n = 4).

GRO- and thrombin up-regulate the same vascular regulatory proteins and growth factors in HUVECs.

View larger version (15K): [in this window] [in a new window]   Figure 3. Immunoblots of CD31, Ang-2, KDR, MMP-1, and MMP-2 following 24 hours of incubation with thrombin (0.5 unit/mL) or GRO- (3 µg/mL) or VEGF (5 ng/mL) at 24 hours in HUVECs. Actin or tubulin were used as loading controls. Representative of two to three experiments.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effect of anti-GRO- antibody or anti-CXCR2 antibody on induction of CD31 (A), KDR (B), Ang-2 (C), CXCR2 (D and E), MMP-1 (F), and MMP-2 (G) in HUVECs. Immunoblots of protein extracted from cells incubated with agonist, anti-GRO- antibody, or irrelevant antibody for 24 hours at 37°C. C, control buffer. Ab, anti-GRO- or anti-CXCR2 (3 µg/mL). TRP, TRAP (100 µmol/L). Ig, irrelevant IgG antibody. Actin was used as loading control. Representative of two to three experiments.

Anti-GRO- antibody or shRNA versus GRO- inhibit the effect of thrombin on up-regulation of MMP-1, MMP-2, CD31, Ang-2, KDR, VEGF, and CXCR2.

The specificity of this reaction was further shown by using an shRNA knockdown of GRO- in 4T1, B16F10, and HUVECs (Fig. 5A ). Successful KD was obtained with cell lines G2, G3, G2/8, and hG2. As found with other tumor cell lines (10, 11), thrombin up-regulated VEGF and Ang-2 mRNA levels in cells transfected with empty vector. Preliminary data using semiquantitative PCR revealed inhibition of thrombin-induced up-regulation of VEGF in G2/G8 and G3 KD 4T1 cells (data not shown).

View larger version (28K): [in this window] [in a new window]   Figure 5. Effect of shRNA GRO- KD in 4T1, B16F10, and HUVEC on thrombin-induced up-regulation of VEGF and Ang-2. A, RT-PCR of GRO- KD 4T1, B16F10 cells and HUVECs with clones G2, G3, G4, G5, G8, G2/G8, hG1, and hG2. GAPDH and hypoxanthine phosphoribosyltransferase (HPRT) and tubulin are used as internal standards. HOH, control without cDNA. B, real-time RT-PCR demonstrating absence of thrombin-induced up-regulation of VEGF and Ang-2 in GRO- KD 4T1 and B16F10 cells. Left: V+T, empty vector + thrombin (0.5 unit/mL for 1 hour at 37°C); G3+T, clone G3+T; G2/8+T, clone G2/G8 + thrombin. Right: V, empty vector; VT, vector + thrombin; SV, scrambled vector; SVT, scrambled vector + thrombin; Kd, knockdown of GRO-; KdT, Kd of GRO- + thrombin (n = 3-6, P < 0.01 for V versus T and SV versus SVT). C, real-time RT-PCR demonstrating inhibition of thrombin-induced up-regulation of ANG-2, CD31, CXCR2, and KDR in GRO- KD HUVECs with clone hG2. Black columns, empty vector + thrombin; white columns, GRO- KD; gray columns, GRO- KD cells treated with thrombin (n = 1-3). D, effect of shRNA GRO- KD on thrombin-induced chemotaxis of 4T1 and B16F10 cells. Thrombin (0.1 unit/mL) was placed in the lower chamber of a Transwell apparatus and 5 x 104 tumor cells were applied to the upper chamber for 3 hours at 37°C. C, buffer; T, buffer + thrombin. Y axis, absorbance of cells stained with crystal violet. The differences between thrombin versus GRO- knockdown were significant at the P < 0.03 and P < 0.01 level for 4T1 and B16F10 cells, respectively (n = 5).

A functional chemotaxis analysis was also used for GRO- KD cells to confirm operational GRO- KD. Figure 5D shows that GRO- KD successfully inhibits thrombin-induced chemotaxis in 4T1 and B16F10 cells. Empty vector had no effect on baseline chemotaxis. Scrambled siRNA had no effect on thrombin-induced chemotaxis.

Effect of GRO- and hirudin on in vivo angiogenesis, tumor growth, seeding, and metastasis. We next designed studies to test whether thrombin-induced up-regulation and secretion of GRO- in tumor cells was contributing to tumor angiogenesis, growth, and metastasis in vivo. GRO- KD 4T1 tumor cells were injected into syngeneic mice to follow tumor angiogenesis, growth, and metastasis. We hypothesized that tumor incapable of synthesizing and secreting GRO- would have fewer blood vessels, smaller tumor nodules, and less metastasis. We also hypothesized that the inhibitory effect of the potent thrombin inhibitor, hirudin, on 4T1 tumor growth (8) would be diminished to absent in GRO- KD tumors. Such proved to be the case. Figure 6A shows decreased 4T1 tumor growth in vivo, which varied from 4- to 3-fold during earlier time points (1-13 days) to 2-fold at later time points (14-25 days). Of particular note is the observation that hirudin had no effect on tumor growth in GRO- KD cells, suggesting that the hirudin-inhibited endogenous thrombin effect was operating through GRO-.

View larger version (72K): [in this window] [in a new window]   Figure 6. In vivo tumor growth and angiogenesis of GRO- KD 4T1 cells in syngeneic wild-type mice with and without inhibition of endogenous thrombin by hirudin. A, BALB/c mice were injected s.c. with 1 x 105 GRO- KD 4T1 cells or empty vector 4T1 cells. Animals were treated with 10 mg/kg hirudin 5 minutes before and 4 hours after tumor injection as well as daily for 10 days, followed by treatment every other day for 7 days. Their tumor volume was then measured (n = 9 for each group). B, immunohistochemistry staining for hotspot VWF-positive vascular endothelial cells. Section from representative control animal with empty vector tumor cells (a) shows higher number and intensity of small vessels compared with animals injected with GRO- KD cells (b), animals treated with empty vector cells plus hirudin (c), or animals injected with GRO- KD cells and treated with hirudin (d), n = 5 for each group.

GRO-

GRO-

GRO-

GRO-

GRO-

GRO-

We next focused our attention on angiogenesis, using real-time RT-PCR for CD105 and VWF in tumor nodules of 4T1 cells as surrogate markers for angiogenesis. Inhibition of tumor nodule CD105 mRNA was observed when using shRNA GRO- KD cells, hirudin alone, or both of 40% (P = 0.02), 36% (P = 0.002), and 51% (P = 0.002), respectively, when compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal control mRNA (n = 3). Similar results were obtained with VWF as marker of 40% (P = 0.02), 36% (P = 0.002), and 55% (P = 0.002), respectively, for GRO-, hirudin alone, or both (n = 3). This was confirmed by immunohistochemistry with the VWF marker (Fig. 6B). Control nodules had higher numbers and intensity of staining than the experimental samples. The angiogenesis index for control nodule (270 ± 33) was decreased 38% in GRO- KD nodules (P = 0.002), 51% with hirudin alone (P = 0.030), and 47% in both (P = 0.042; n = 5). It should be pointed out that functional antiangiogenesis effects may also occur without or with minimal reduction in increased density, as discussed by Hlatky et al. (29).

Thrombin up-regulation of vascular genes is different from bFGF or VEGF. To examine the specificity of GRO- up-regulation by thrombin/TRAP, we analyzed the effect of two other vascular growth factors, bFGF and VEGF, on GRO- and angiogenesis. bFGF (1-2 µg/filter disc on egg) enhanced angiogenesis 2.6-fold at 72 hours (n = 7, P < 0.001) in the CAM assay. However, bFGF had no effect on GRO- up-regulation by immunoblot in HUVECs, yet it up-regulated VEGF, KDR, CD31, CXCR2, and Ang-2 (data not shown). VEGF similarly had no effect on GRO-, yet it up-regulated CD31 and Ang-2 in HUVEC and had no effect on KDR (Fig. 3) or CXCR2 (data not shown). Thus, thrombin-induced angiogenesis specifically requires the up-regulation of GRO-.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
These data clearly show a pivotal role for the chemokine GRO- and its endothelial cell CXCR2 receptor in thrombin-induced neoangiogenesis. This was documented by analyzing the effect of both thrombin and GRO- on the up-regulation of vascular regulatory genes as well as angiogenesis in the CAM, endothelial cell cord–forming, and endothelial growth assays. Anti-GRO- antibody inhibited both GRO- as well as thrombin-induced angiogenesis in the CAM, endothelial cell cord formation, and endothelial cell growth assays.

Both thrombin and GRO- up-regulate MMP-1, MMP-2, VEGF, KDR, Ang-2, and CD31 and thrombin up-regulates the GRO- receptor CXCR2. Up-regulation of MMP-1 (20), VEGF (10), KDR (14, 30), and Ang-2 (11) has been reported for thrombin but not for GRO-. Up-regulation of the GRO- receptor CXCR2 has not been reported for thrombin or GRO-. Up-regulation of GRO- by thrombin has been reported for HUVEC (31). Our data indicate that this is likely to be a general response because it is also up-regulated by thrombin in several tumor cell lines, murine B16F10 and UMCL, and human MCF7 breast and PC3 prostate cell lines.

The similar pattern of up-regulation of these vascular regulatory genes in HUVEC and tumor cells by both thrombin and GRO- as agonists, as well as the marked thrombin-induced up-regulation of GRO-, suggested that thrombin could be having its effect through the up-regulation of GRO-. Such proved to be the case because (a) anti-GRO- antibody inhibited thrombin-induced angiogenesis in both the CAM and endothelial cell cord–forming assay; (b) anti-GRO- antibody inhibited thrombin-induced up-regulation of MMP-1, MMP-2, VEGF, KDR, Ang-2, CD31, and CXCR2; (c) VEGF, Ang-2, KDR, and CXCR2 were not up-regulated by thrombin in GRO- KD cells. Further proof that thrombin is operating through GRO- ligation of its CXCR2 receptor was obtained by demonstrating that an antibody against CXCR2 also inhibited the effect of thrombin on up-regulation of the vascular regulatory genes tested.

Thrombin-induced up-regulation of GRO- in tumor cells is likely to contribute to the effect of thrombin on endothelial cells by providing an additional source of GRO- production. This is supported by in vivo studies on endogenous thrombin and tumor growth in wild-type mice, injected with GRO- KD 4T1 cells in the presence and absence of hirudin. Hirudin had no effect on the impaired tumor growth of GRO- KD cells. Thus, tumor cells can then be contributing to angiogenesis by the production of GRO- as is the case with VEGF (10), and thrombin can be contributing to the malignant tumor phenotype by enhancing tumor angiogenesis. Indeed, it has been reported that angiogenesis contributes to the activation of dormant tumor cells (32).

The mechanism of thrombin-induced up-regulation of angiogenesis (in association with the seven vascular regulatory genes tested) operates through the ligation of its PAR-1 receptor. This was documented with the protein-activated receptor agonist SFLLRNPNDKYEPF whose gene up-regulation effect was also inhibited by anti-GRO- antibody. MMP-1 has similarly been reported to be up-regulated by ligation of PAR-1 in human endothelial cells (20).

The requirement of GRO- up-regulation for angiogenesis is specific for thrombin. The angiogenic growth factor bFGF does not up-regulate GRO-, whereas bFGF up-regulates KDR, Ang-2, CXCR2, and CD31 in HUVEC. VEGF does not up-regulate GRO- or KDR in HUVEC, whereas it up-regulates Ang-2 and CD31 in HUVEC. In addition, we have recently observed that thrombin-induced up-regulation of cathepsin D and -synuclein does not require GRO-,⁴ further supporting GRO- specificity for thrombin-induced angiogenesis.

Stimulation of angiogenesis by GRO- has been suggested from histochemical studies by the inhibitory effect of anti-GRO- antibody on angiogenesis associated with DU145 prostate tumor growth in severe combined immunodeficient mice as well as the use of tumor homogenates applied to a rat corneal micropocket assay, before and after in vivo treatment of mice with anti-GRO antibody (27). It also has been suggested by experiments in which GRO- has been transfected into a squamous cell carcinoma cell line with low levels of GRO-, which resulted in increased CD31⁺ blood vessels as well as tumor growth (22). Our work provides data for the first direct evidence of GRO--induced angiogenesis as documented by the CAM, endothelial cord formation, and endothelial growth assays as well as the up-regulation of seven vascular regulatory proteins and growth factors. Our in vivo studies provide further evidence that both GRO- and thrombin are capable of regulating tumor angiogenesis. Stimulation of angiogenesis by thrombin is well documented in the CAM and endothelial cell cord formation assays (12, 15). Our data report the first evidence for the pivotal role of GRO- in thrombin-induced angiogenesis in HUVEC as well as tumor cells.

Exclusive of its effect on angiogenesis, both thrombin (2, 4, 5, 7, 8) and GRO- are pro-oncogenic (22, 23, 27, 33). GRO- is an autocrine growth factor first identified in melanoma. Overexpression in immortalized mouse melanocytes enabled the cells to form large colonies in soft agar as well as tumors in nude mice (33). In melanoma, GRO- has been shown to have mitogenic as well as angiogenic effects on tumor growth (33). A DU145 human prostate cancer cell line had in vivo growth, which correlated with tumor-derived GRO- and could be inhibited with anti-GRO- antibody (31). GRO- promotes murine squamous cell carcinoma growth, metastasis, and leukocyte infiltration by a host CXCR2-dependent mechanism. An autocrine increase in growth was also noted in vivo (22). Variants of murine squamous cell carcinoma that grow and metastasize more rapidly in vivo express increased levels of GRO- compared with those that grow and metastasize more slowly (22). Transfection of GRO- into a cell line with low levels of GRO- showed an increased rate of growth and spontaneous metastasis in BALB/c mice in association with increased infiltration of host leukocytes. This increase in growth was inhibited in CXCR2(–/–) mice (22). S.c. growth of Lewis lung carcinoma as well as spontaneous pulmonary metastasis is significantly decreased in CXCR2–/– mice; however, this was attributed to decreased angiogenesis because GRO- had no effect on Lewis lung carcinoma in vitro (23).

The above data clearly describe a new mechanism for thrombin induction of the malignant phenotype in numerous tumor cell lines. Its effect on tumor angiogenesis requires up-regulation of the pro-oncogenic/angiogenic gene GRO-. Thus, thrombin serves a dual purpose in enhancing angiogenesis as well as tumor growth and metastasis.

	Acknowledgments

 
Grant support: NIH grant HL 13336 and grants from the Hildegarde D. Becher Foundation, Helen Polansky Research Fund, and the Dorothy and Seymour Weinstein Research Fund.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Presented at the 47^th Annual Meeting of the American Society of Hematology, December 12, 2005.

	Footnotes

 
Note: M. Caunt and L. Hu contributed equally to this work.

⁴ L. Hu and S. Karpatkin, unpublished data.

Received 7/21/05. Revised 2/ 1/06. Accepted 2/15/06.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Nierodzik M, Plotkin A, Kajumo F, Karpatkin S. Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo. J Clin Invest 1991;87:229–36.[Medline]
2. Nierodzik M, Kajumo F, Karpatkin S. Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and metastasis in vivo. Cancer Res 1992;52:3267–72.[Abstract/Free Full Text]
3. Nierodzik M, Bain R, Liu LX, et al. Presence of the seven transmembrane thrombin receptor on human tumor cells: effect of activation on tumor adhesion to platelets and tumor tyrosine phosphorylation. Br J Haematol 1996;92:452–7.[CrossRef][Medline]
4. Klepfish A, Greco MA, Karpatkin S. Thrombin stimulates melanoma tumor-cell binding to endothelial cells and subendothelial matrix. Int J Cancer 1993;53:978–82.[Medline]
5. Zain J, Huang YQ, Feng X, et al. Concentration-dependent dual effect of thrombin on impaired growth/apoptosis or mitogenesis in tumor cells. Blood 2000;95:3133–8.[Abstract/Free Full Text]
6. Camerer E, Qazi A, Duong D, et al. Platelets, protease-activated receptors, and fibrinogen in hematogenous metastasis. Blood 2004;104:397–401.[Abstract/Free Full Text]
7. Nierodzik M, Chen K, Takeshita K, et al. Protease-activated receptor 1 (PAR-1) is required and rate-limiting for thrombin-enhanced experimental pulmonary metastasis. Blood 1998;92:3694–700.[Abstract/Free Full Text]
8. Hu L, Lee M, Campbell W, Perez-Soler R, Karpatkin S. Role of endogenous thrombin in tumor implantation, seeding and spontaneous metastasis. Blood 2004;104:2746–51.[Abstract/Free Full Text]
9. Wojtukiewicz M, Tang D, Ciarelli J, et al. Thrombin increases the metastatic potential of tumor cells. Int J Cancer 1993;54:793–806.[Medline]
10. Huang Y-Q, Li J-J, Hu L, Lee M, Karpatkin S. Thrombin induces increased expression and secretion of VEGF from human FS4 fibroblasts, DU145 prostate cells and CHRF megakaryocytes. Thromb Haemost 2001;86:1094–8.[Medline]
11. Huang Y-Q, Hu L, Lee M, Karpatkin S. Thrombin induces increased expression and secretion of angiopoietin-2 from human umbilical vein endothelial cells. Blood 2002;99:1646–50.[Abstract/Free Full Text]
12. Haralabopoulos G, Grant D, Kleinman H, Maragoudakis M. Thrombin promotes endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo. Am J Physiol 1997;273:239–45.
13. Herbert JM, Dupuy E, Laplace MC, Zini JM, Bar Shavit R. Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway. Biochem J 1994;303:227–31.
14. Tsopanoglou NE, Maragoudakis ME. On the mechanism of thrombin-induced angiogenesis. Potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors. J Biol Chem 1999;274:23969–76.[Abstract/Free Full Text]
15. Caunt M, Huang Y, Brooks P, Karpatkin S. Thrombin induces neoangiogenesis in the chick chorioallantoic membrane. J Thromb Haemost 2003;1:2097–102.[CrossRef][Medline]
16. Ollivier V, Chabbat J, Herbert JM, Hakim J, de Prost D. Vascular endothelial growth factor production by fibroblasts in response to factor VIIa binding to tissue factor involves thrombin and factor Xa. Arterioscler Thromb Vasc Biol 2000;20:1374–81.[Abstract/Free Full Text]
17. Moloney JP, Sillimon CC, Ambruso DR, et al. In vitro release of VEGF during platelet aggregation. Am J Physiol 1988;275:H1054.
18. Mohle R, Green D, Moore M, Nachman R, Rafii S. Constitutive production and thrombin-induced release of VEGF by human megakaryocytes and platelets. Proc Natl Acad Sci U S A 1997;94:663–8.[Abstract/Free Full Text]
19. Li JJ, Huang YQ, Basch R, Karpatkin S. Thrombin induces the release of angiopoietin-1 from platelets. Thromb Haemost 2001;85:204–6.[Medline]
20. Duhamel-Clerin E, Orvain C, Lanza F, Cazenave JP, Klein-Soyer C. Thrombin receptor-mediated increase of two matrix metalloproteinases, MMP-1 and MMP-3, in human endothelial cells. Arterioscler Thromb Vasc Biol 1997;17:1931–8.[Abstract/Free Full Text]
21. Baggiolini M, DeWald B, Moser B. Interleukin-8 and related chemotactic cytokines-CXC and CC chemokines. Adv Immunol 1994;55:97–179.[Medline]
22. Loukinova E, Dong G, Enamorado-Ayalya I, et al. Growth regulated oncogene- expression by murine squamous cell carcinoma promotes tumor growth, metastasis, leukocyte infiltration and angiogenesis by a host CXC Receptor-2 dependent mechanism. Oncogene 2000;19:3477–86.[CrossRef][Medline]
23. Keane M, Belperio J, Xue Y, Burdick M, Strieter R. Depletion of CXCR2 inhibits tumor growth and angiogenesis in a murine model of lung cancer. J Immunol 2004;172:2853–60.[Abstract/Free Full Text]
24. Strieter RM, Burdick BM, Gomports BN, Belperio JA, Keane MP. C-X-C chemokines in angiogenesis. Cytokine Growth Factor Rev 2005;16:593–609.[CrossRef][Medline]
25. Devalaraja R, Nanney L, Du J, et al. Delayed wound healing in CXCR2 knockout mice. J Invest Dermatol 2000;115:234–44.[CrossRef][Medline]
26. Loukinova E, Chen Z, Van Waes C, Dong G. Expression and proangiogenesis chemokine GRO 1 in low and high metastasis variants of PAM murine squamous cell carcinoma is differently regulated by IL-1, EGF and TGFß1 through NF-B dependent and independent mechanisms. Int J Cancer 2001;94:637–44.[CrossRef][Medline]
27. Moore B, Arenberg D, Stoy K, et al. Distinct CXC chemokines mediate tumorigenicity of prostate cancer cells. Am J Pathol 1999;154:1503–12.[Abstract/Free Full Text]
28. Smith C, Chen Z, Dong G, et al. The host environment promotes the development of primary and metastatic squamous cell carcinomas that constitutively express proinflammatory cytokines IL-1a, IL-6, GM-CSF, and KC. Clin Exp Metastasis 1998;16:655–64.[CrossRef][Medline]
29. Hlatky L, Hahnfeldt P, Folkman J. Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us. J Natl Cancer Inst 2002;94:883–93.[Free Full Text]
30. Wang J, Morita I, Onodera M, Murota S-I. Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide. J Cell Physiol 2002;190:238–50.[CrossRef][Medline]
31. Murakami K, Ueno A, Yamanouchi K, Kondo T. Thrombin induces GRO-/MGSA production in human umbilical vein endothelial cells. Thromb Res 1995;79:387–94.[CrossRef][Medline]
32. Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989;339:58–61.[CrossRef][Medline]
33. Luan J, Shattuck-Brandt R, Haghnegahdar H, et al. Mechanism and biological significance of constitutive expression of MGSA/GRO chemokines in malignant melanoma tumor progression. J Leukoc Biol 1997;62:588–97.[Abstract]

This article has been cited by other articles:

				W. Yan and X. Chen Identification of GRO1 as a Critical Determinant for Mutant p53 Gain of Function J. Biol. Chem., May 1, 2009; 284(18): 12178 - 12187. [Abstract] [Full Text] [PDF]

				L. Hu, S. Ibrahim, C. Liu, J. Skaar, M. Pagano, and S. Karpatkin Thrombin Induces Tumor Cell Cycle Activation and Spontaneous Growth by Down-regulation of p27Kip1, in Association with the Up-regulation of Skp2 and MiR-222 Cancer Res., April 15, 2009; 69(8): 3374 - 3381. [Abstract] [Full Text] [PDF]

				P. Zania, D. Gourni, A. C. Aplin, R. F. Nicosia, C. S. Flordellis, M. E. Maragoudakis, and N. E. Tsopanoglou Parstatin, the Cleaved Peptide on Proteinase-Activated Receptor 1 Activation, Is a Potent Inhibitor of Angiogenesis J. Pharmacol. Exp. Ther., February 1, 2009; 328(2): 378 - 389. [Abstract] [Full Text] [PDF]

				L. Hu, J. M. Roth, P. Brooks, J. Luty, and S. Karpatkin Thrombin Up-regulates Cathepsin D which Enhances Angiogenesis, Growth, and Metastasis Cancer Res., June 15, 2008; 68(12): 4666 - 4673. [Abstract] [Full Text] [PDF]

				L. Hu, J. M. Roth, P. Brooks, S. Ibrahim, and S. Karpatkin Twist Is Required for Thrombin-Induced Tumor Angiogenesis and Growth Cancer Res., June 1, 2008; 68(11): 4296 - 4302. [Abstract] [Full Text] [PDF]

				H. Kawanishi, Y. Matsui, M. Ito, J. Watanabe, T. Takahashi, K. Nishizawa, H. Nishiyama, T. Kamoto, Y. Mikami, Y. Tanaka, et al. Secreted CXCL1 Is a Potential Mediator and Marker of the Tumor Invasion of Bladder Cancer Clin. Cancer Res., May 1, 2008; 14(9): 2579 - 2587. [Abstract] [Full Text] [PDF]

				J.-S. Silvestre, Z. Mallat, A. Tedgui, and B. I. Levy Post-ischaemic neovascularization and inflammation Cardiovasc Res, May 1, 2008; 78(2): 242 - 249. [Abstract] [Full Text] [PDF]

				M. Hsu, S.-Y. Wu, S.-S. Chang, I.-J. Su, C.-H. Tsai, S.-J. Lai, A.-L. Shiau, K. Takada, and Y. Chang Epstein-Barr Virus Lytic Transactivator Zta Enhances Chemotactic Activity through Induction of Interleukin-8 in Nasopharyngeal Carcinoma Cells J. Virol., April 1, 2008; 82(7): 3679 - 3688. [Abstract] [Full Text] [PDF]

				H. H. Versteeg, F. Schaffner, M. Kerver, H. H. Petersen, J. Ahamed, B. Felding-Habermann, Y. Takada, B. M. Mueller, and W. Ruf Inhibition of tissue factor signaling suppresses tumor growth Blood, January 1, 2008; 111(1): 190 - 199. [Abstract] [Full Text] [PDF]

				C. Bechara, X. Wang, H. Chai, P. H. Lin, Q. Yao, and C. Chen Growth-related oncogene-{alpha} induces endothelial dysfunction through oxidative stress and downregulation of eNOS in porcine coronary arteries Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3088 - H3095. [Abstract] [Full Text] [PDF]

				H. Uusitalo-Jarvinen, T. Kurokawa, B. M. Mueller, P. Andrade-Gordon, M. Friedlander, and W. Ruf Role of Protease Activated Receptor 1 and 2 Signaling in Hypoxia-Induced Angiogenesis Arterioscler. Thromb. Vasc. Biol., June 1, 2007; 27(6): 1456 - 1462. [Abstract] [Full Text] [PDF]

				S. Karpatkin MCP-1 muscles in on pericytes Blood, February 1, 2007; 109(3): 849 - 850. [Full Text] [PDF]

				J. Ma, Q. Wang, T. Fei, J.-D. J. Han, and Y.-G. Chen MCP-1 mediates TGF-{beta}-induced angiogenesis by stimulating vascular smooth muscle cell migration Blood, February 1, 2007; 109(3): 987 - 994. [Abstract] [Full Text] [PDF]

				J. Sevastos, S. E. Kennedy, D. R. Davis, M. Sam, P. W. Peake, J. A. Charlesworth, N. Mackman, and J. H. Erlich Tissue factor deficiency and PAR-1 deficiency are protective against renal ischemia reperfusion injury Blood, January 15, 2007; 109(2): 577 - 583. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Caunt, M. Articles by Karpatkin, S. Search for Related Content PubMed PubMed Citation Articles by Caunt, M. Articles by Karpatkin, S.