Affiliations of authors: J. R. Merchan, Division of Hematology-Oncology, Department of Medicine and the Center for Study of the Tumor Microenvironment, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, and The Clinical Investigator Training Program, Beth Israel Deaconess Medical CenterHarvard/Massachusetts Institute of Technology Health Sciences and Technology, in collaboration with Pfizer Inc.; B. Chan, S. Kale, Division of Nephrology, Department of Medicine and the Center for Study of the Tumor Microenvironment, Beth Israel Deaconess Medical Center and Harvard Medical School; L. E. Schnipper, Division of Hematology-Oncology, Department of Medicine and the Center for Study of the Tumor Microenvironment, Beth Israel Deaconess Medical Center and Harvard Medical School; V. P. Sukhatme, Divisions of Hematology-Oncology and Nephrology, Department of Medicine and the Center for Study of the Tumor Microenvironment, Beth Israel Deaconess Medical Center and Harvard Medical School.
Correspondence to: Vikas P. Sukhatme, M.D., Ph.D., Beth Israel Deaconess Medical Center, 330 Brookline Ave., Dana 517, Boston, MA 02215 (e-mail: vsukhatm{at}caregroup.harvard.edu).
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ABSTRACT |
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INTRODUCTION |
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Some reports (20, 21) have suggested that proteases may stimulate tumor progression and angiogenesis. However, recent studies have shown that some of these proteases, in fact, may have a protective effect against tumor progression. First, mice deficient in plasminogen activator inhibitor 1 (PAI-1, the natural inhibitor of plasminogen activators) did not support invasion or angiogenesis of implanted tumors (22). However, PAI-1 may have a dual role in angiogenesis, as an activator or an inhibitor, depending on the tumors microenvironment and its level of PAI-1 (2325). Second, when tumors were implanted in integrin 1-deficient mice (which express higher levels of matrix metalloproteinases and plasma angiostatin), reduced tumor angiogenesis was observed (26). Plasma from these mice inhibited endothelial cell proliferation, and this inhibition was decreased by administering an angiostatin-neutralizing antibody or matrix metalloproteinase inhibitors (26). Third, mice injected with colon cancer cells that overexpressed t-PA had a markedly lower number of liver metastases and a higher survival rate than mice injected with untransfected colon cancer cells (27). A study of tumor specimens taken from patients with melanoma (28) or with breast cancer (29) showed that tumors expressing a high level of t-PA were associated with better prognosis. Thus, it has been hypothesized that human plasma may serve as a rich source of biologically important angiogenesis inhibitors (15). Increasing the antiangiogenic activity of plasma proteins by proteolytic cleavage may alter the behavior of a malignant tumor and could have clinically useful implications.
In this article, we investigate whether the antiangiogenic activity of human plasma can be increased by treatment with recombinant t-PA (rt-PA) and captopril in vitro and in vivo. We use an in vivo angiogenesis assay in mice to determine whether treatment with rt-PA and captopril decreases angiogenesis in Matrigel plugs, and we measure the antiangiogenic activity in the plasma of cancer patients treated with rt-PA and captopril. Finally, we investigate whether the observed antiangiogenic effects were dependent on angiostatin.
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MATERIALS AND METHODS |
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rt-PA (Genentech, San Francisco, CA) and captopril (Sigma-Aldrich, St. Louis, MO) were diluted in sterile phosphate-buffered saline (PBS) and used for the bioassays. Heparin (Elkins-Sinn, Cherry Hill, NJ) at 1 U/mL or lepirudin (Aventis Pharmaceuticals, Kansas City, MO) at 5 µg/mL was added to fresh frozen plasma (FFP) or patients plasma to prevent clot formation. Matrigel (Collaborative Biomedical Products, Bedford, MA), a basement membrane preparation from the Engelbreth-Holm-Swarm mouse sarcoma, was used at 7 mg/mL for in vitro angiogenesis (endothelial cell tube formation) assays and at 10 mg/mL for in vivo angiogenesis (Matrigel plug) assays (see below). Basic fibroblast growth factor (bFGF) was purchased from PeproTech, Rocky Hill, NJ. The cell proliferation reagent WST-1 (Roche, Indianapolis, IN) was used for proliferation assays. WST-1 is a tetrazolium salt that is cleaved to formazan by mitochondrial dehydrogenases in viable cells. A murine monoclonal antibody against human angiostatin (Calbiochem, San Diego, CA) was used for western blotting and immunodepletion. A rabbit polyclonal antibody against mouse angiostatin (Affinity BioReagents, Golden, CO) that cross-reacts with human angiostatin was used for western blotting. Human angiostatin (kringles 14) was obtained from Calbiochem.
Concentrations of rt-PA and Captopril Used
Pharmacokinetic studies have shown that captopril concentrations of 0.11 µM are achieved in the plasma after captopril doses of 2537.5 mg three times a day (30) and that plasma rt-PA concentrations in the range of 0.51.8 µg/mL are achieved in healthy volunteers and cardiac patients after rt-PA doses of 0.004 mg/kg/min for up to 90 minutes (31, 32). We used captopril and rt-PA concentrations in these dose ranges.
Case Reports
Patient 1 was a 46-year-old woman with a history of metastatic malignant fibrous histiocytoma. She had multiple recurrences after surgical resections (pulmonary, hepatic, and subcutaneous nodules), radiation therapy, and thalidomide treatment, and she had refused standard chemotherapy. She was screened for bleeding disorders and for brain metastases and gave written informed consent. She first received 25 mg of captopril by mouth three times a day, and then 1 week later, she received the first of four 12-hour intravenous infusions of rt-PA over a 4-week period with increasing doses each week, from 0.015 mg/kg/h (first dose), to 0.02 mg/kg/h, to 0.03 mg/kg/h, to 0.035 mg/kg/h. Blood was taken for coagulation tests (thrombin, prothrombin, and activated partial thromboplastin times and fibrinogen level) and for bioassays at various times before, during, and after the infusion. Similarly, two additional patients (patients 2 and 3) with advanced melanoma were treated (as part of an Institutional Review Board-approved phase I trial; both provided written informed consent) with captopril at 37.5 mg by mouth three times a day, and 1 week later, they received a 12-hour infusion of rt-PA at a dose of 0.015 mg/kg/h. No patient experienced a substantial adverse reaction during or after the infusions.
Human Plasma
Outdated FFP was obtained from Beth Israel Deaconess Medical Centers blood bank. Blood was collected from patients with cancer from a peripheral vein into citrated tubes. The blood was immediately centrifuged at 3210g for 10 minutes. Both FFP and the patients plasma were filter sterilized (0.2-µm [pore size] sterile filters; Millipore, Bedford, MA) and then stored at -20 °C for future use. For treated FFP, captopril (1 µM) and rt-PA (10 µg/mL) were added to 1 mL of FFP and incubated for 3 hours at 37 °C before use.
Cell Culture
Human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells from the lung (HMVEC-Ls) were obtained from Clontech Laboratories (Palo Alto, CA) and used between passages 3 and 5. They were maintained in EGM2-MV (full endothelial cell growth) medium (Clontech) that contained endothelial basal medium 2 (EBM-2), supplemented with 5% fetal bovine serum (FBS), gentamicin, amphotericin B, hydrocortisone, ascorbic acid, vascular endothelial growth factor (VEGF), bFGF, human epidermal growth factor, and insulin-like growth factor I. Human renal epithelial (HRE) cells and primary human fibroblasts (IMR-90) were used for specificity assays. HRE cells were maintained in renal epithelial cell growth medium (renal epithelial cell basal medium, supplemented with 0.5% FBS, insulin, hydrocortisone, epinephrine, tri-iodothyronine, transferrin, human epidermal growth factor, gentamicin, and amphotericin B; Clontech). IMR-90 cells were maintained in Dulbeccos modified Eagle medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL). Cells were grown at 37 °C in a 100% humidified incubator with an atmosphere of 5% CO2/95% air. When the cell cultures were 80%90% confluent, cells were harvested with trypsin and resuspended to the cell density required for each assay.
Matrigel Tube Formation Assay
Each well of prechilled 48-well cell culture plates was coated with 100 µL of unpolymerized Matrigel (7 mg/mL) and incubated at 37 °C for 3045 minutes. HUVECs were harvested with trypsin, and 4 x 104 cells were resuspended in 300 µL of full endothelial cell growth medium and treated with the various agents before plating onto the Matrigel-coated plates. After 12 hours of incubation, endothelial cell tube formation was assessed with an inverted photomicroscope (Nikon, Melville, NY). Microphotographs of the center of each well at low power (x40) were taken with a SPOT camera (Diagnostic Instruments, Inc., Sterling Heights, MI) with the aid of imaging-capture software (Compix Inc. Imaging Systems, Cranberry Township, PA). Tube formation in the microphotographs was quantitatively analyzed (total tube length) with Simple PCI imaging analysis software (Compix). Tube formation by untreated HUVECs in full endothelial cell growth medium was used as a negative control, and tube formation in cultures treated with actinomycin D (Sigma-Aldrich) at 7.5 µg/mL was used as a positive (inhibitory) control.
Cell Proliferation Assay
A total of 4 x 103 cells in 100 µL of the appropriate basal medium with 1% FBS, penicillin (100 U/mL), and streptomycin (100 µg/mL) was placed into each well of a 96-well plate, treated with the test agents, and incubated at 37 °C for 72 hours; control cells were cultured in basal medium, 1% FBS, and antibiotics, as above. Because we observed that plasma was a potent stimulant of endothelial cell proliferation, no additional proliferation stimulus (VEGF or bFGF) was added. After the 72-hour incubation, WST-1 (10 µL) was added to each well, and after a 3-hour incubation at 37 °C, absorbance at 450 nm was determined for each well with a microplate reader (Bio-Rad Laboratories, Hercules, CA). Data presented are the average of triplicate experiments.
In Vivo Angiogenesis (Matrigel Plug) Assay
The Matrigel plug assay was performed as previously described (33, 34) with the following modifications. Five- to six-week-old male C57/BL6 mice (The Jackson Laboratory, Bar Harbor, ME) were used. Unpolymerized Matrigel (0.5 mL) supplemented with bFGF (500 ng/mL) was injected subcutaneously in the left lower abdominal wall for the stimulated control and treatment groups, and 0.5 mL of Matrigel mixed with a volume of sterile PBS equivalent to that of bFGF was injected for the unstimulated (no bFGF) control group. Each mouse had one plug. Three mice per group were treated for 10 days with 1) rt-PA (60 µg diluted in 100 µL of 1x sterile PBS per day, subcutaneously) and captopril (150 µg diluted in 100 µL of 1x sterile PBS per day, intraperitoneally); 2) rt-PA alone subcutaneously, with a volume of PBS equivalent to that of captopril intraperitoneally; 3) captopril (150 µg) intraperitoneally, with a volume of PBS equivalent to that of rt-PA subcutaneously; or 4) volumes of PBS equivalent to those of rt-PA and captopril, subcutaneously and intraperitoneally, respectively. Mice were killed on day 10, and the Matrigel plugs were removed, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sections were examined by light microscopy, and the total number of microvessels containing red blood cells in 10 high-power fields (x400 magnification) was counted in a blinded fashion. Only microvessels that contained red blood cells were counted. Results shown represent the average of counts from three Matrigel plugs per group and 95% confidence intervals (CIs). All animal studies were reviewed and approved by the animal care and use committee of Beth Israel Deaconess Medical Center and were in accordance with the guidelines of the Department of Health and Human Services.
Affinity Chromatography
LysineSepharose (Pharmacia, Peapack, NJ) chromatography was used to separate angiostatin from the other proteins in treated FFP. Briefly, a lysineSepharose column (6 mL) was made as described by the manufacturer. The procedure was performed at 4 °C. Treated FFP (10 mL) was loaded onto the column pre-equilibrated with 50 mM sodium phosphate (pH 7.5), and then the column was washed successively with 10 volumes of 50 mM sodium phosphate (pH 7.5), five volumes of PBS, and five volumes of 0.5 M NaCl. Retained proteins were eluted with 200 mM -aminocaproic acid (Sigma-Aldrich) in water. The eluted protein was dialyzed (dialysis membrane with a molecular weight cutoff of 3000; Pierce, Rockford, IL) against 4 L of PBS for 48 hours, concentrated to the original volume of the plasma, filter sterilized, and stored at -20 °C for future use.
Angiostatin Immunoprecipitation of FFP
Treated FFP (200 µL) was incubated with rocking at 4 °C overnight with a monoclonal antibody against human angiostatin (32 µg/mL). The next day, 50 µL of protein A/G PLUS agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added, and the mixture was rocked for 2 hours at 4 °C and centrifuged (11 750g for 5 minutes). The supernatant (immunoprecipitated plasma) was stored at 20 °C for future use.
Fractionation of Treated FFP
Small-scale anion-exchange chromatographic steps were initially used to optimize the separation of angiostatin from other components in treated FFP with antiangiogenic activity, as follows. Treated FFP (1 mL) was exchanged into buffer A (10 mM TrisHCl [pH 7.4])/50 mM NaCl by use of a NAP-10 column (Pharmacia), and the sample was applied to a Q-Sepharose column (1-mL HiTrap QXL (Pharmacia) pre-equilibrated with buffer A/50 mM NaCl at a rate of 1 mL/min. The column was washed with buffer A until the absorbance at 280 nm returned to baseline. Proteins were eluted with a step gradient of NaCl in 50-mM increments until 500 mM NaCl was reached. The column was then washed with buffer A/1 M NaCl. All fractions were concentrated and exchanged into 1x PBS before testing for antiangiogenic activity. Components with antiangiogenic activity were eluted between 300 mM NaCl and 400 mM NaCl. Preparative scale separation was performed by applying treated FFP to a 20-mL HiPrep 16/10 QXL column (Pharmacia). The column was washed extensively with buffer A/300 mM NaCl. Absorbed proteins were eluted from the column sequentially with buffer A/400 mM NaCl and buffer A/1 M NaCl. All fractions were concentrated, exchanged into 1x PBS, and stored at 20 °C for further use.
Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis (SDSPAGE) and Western Blot Analysis
Protein samples diluted with 1x SDS (Boston BioProducts, Inc., Ashland, MA)/40 mM dithiothreitol were separated by SDSPAGE in 4%20% gels (pre-cast gels; Bio-Rad), followed by electroblotting onto a polyvinylidenedifluoride (PVDF) membrane. After blocking with 2% bovine serum albumin in Tris-buffered saline/Tween 20 (TTBS: 100 mM Tris, 100 mM NaCl, and 0.1% Tween-20) for 1 hour, the PVDF membrane was incubated overnight with the polyclonal angiostatin antibody (2 µg/mL). After washing with TTBS, the membrane was incubated with a horseradish peroxidase-conjugated secondary antibody (Amersham, Arlington Heights, IL; 1 : 5000 dilution) for 1 hour. The protein bands were detected by use of SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Statistical Analysis
Means were compared by use of a Students t test analysis (data analyzed met all criteria for Students t test). The results of the counts for vascular density and cell proliferation are expressed as means with 95% CIs. Differences were considered statistically significant at P<.05. All statistical tests were two-sided.
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RESULTS |
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In vitro angiogenesis was assessed by endothelial cell tube formation and endothelial cell proliferation. To assess tube formation, 4 x 104 HUVECs in the presence of untreated or treated FFP and full endothelial cell growth medium (EGM2-MV) were cultured on Matrigel-coated plates. HUVECs in full endothelial cell growth medium but without FFP were used as a negative control. After 1216 hours of incubation, cultures containing treated FFP had less angiogenesis than cultures containing untreated FFP, as demonstrated by inhibition of endothelial cell tube formation (Fig. 1, CE, and quantitative analysis, J). Angiogenesis of cultures incubated with rt-PA or captopril alone or in combination (Fig. 1
, FH), in the absence of plasma, was equivalent to that in the control cultures. Angiogenesis of cultures increased as the concentration of treated FFP was reduced from 20% to 1% (Fig. 1
, CE). Similar data were obtained when plasma from several patients with cancer was treated in vitro with rt-PA or urokinase and captopril and added to cultures of HUVECs (data not shown).
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Antiangiogenic activity of plasma in vivo was assessed in mice with the Matrigel plug assay. Mice were injected with Matrigel and then treated with rt-PA and captopril. The Matrigel plugs of all groups of mice (except the unstimulated control) contained bFGF as a proangiogenic stimulant. None of the groups developed treatment-associated adverse events. Ten days after Matrigel injection mice were killed, and the plugs were analyzed for angiogenesis by measuring the number of endothelial microvessels formed that contained red blood cells. Mice treated with both rt-PA and captopril had substantially lower angiogenesis (i.e., fewer microvessels; 49 microvessels/10 high-power fields, 95% CI = 33 to 65) than those treated with PBS (93 microvessels/10 high-power fields, 95% CI = 83 to 103), rt-PA alone (67 microvessels/10 high-power fields, 95% CI = 58 to 76), or captopril alone (84 microvessels/10 high-power fields, 95% CI = 79 to 89) (Fig. 3, A).
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Plasma from cancer patient 1 who was treated with captopril and four sequential 12-hour infusions of rt-PA at 0.015, 0.02, 0.03, and 0.035 mg/kg/h was assessed for antiangiogenic activity by its effect on endothelial cell Matrigel tube formation and on cell proliferation. The plasma obtained from patient 1 during infusions of rt-PA at 0.02, 0.03, and 0.035 mg/kg/h had substantial antiangiogenic activity in the HUVEC tube formation assay (Fig. 4, C), and the antiangiogenic activity was slightly higher in plasma obtained during the infusions with higher concentrations of rt-PA (45%, 46%, and 53% inhibition of tube formation from dose levels 2, 3, and 4, respectively, compared with that by pretreatment plasma (control) [Fig. 4
, BE, and F for quantification]). Plasma obtained from patient 1 during the 12-hour infusion also statistically significantly inhibited HUVEC proliferation (67% inhibition; P = .01, two-sided Students t test) compared with plasma obtained from patient 1 before treatment (Fig. 4
, G). This effect lasted for up to 48 hours after the start of the infusion and gradually trended toward baseline over the ensuing 6 days.
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Angiostatin is generated when plasminogen is incubated with plasminogen activators and sulfhydryl donors (17). To determine the contribution of angiostatin to the antiangiogenic effects observed with treated FFP, human angiostatin and treated FFP were tested in Matrigel tube formation assays. Medium containing 10% treated FFP (Fig. 1, D) should contain angiostatin at approximately 10 µg/mL, if full conversion of plasminogen (200 µg/mL in 100% plasma) to angiostatin is assumed. Angiostatin at 10 µg/mL (Fig. 6
, A) or at 50 µg/mL (data not shown) essentially did not inhibit tube formation, but 10% treated FFP strongly inhibited tube formation.
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To confirm this observation, treated FFP was immunodepleted of angiostatin with monoclonal antibodies against human angiostatin, as shown by western blot analysis (Fig. 6, E, lane 5). The antiangiogenic activity of angiostatin-immunodepleted treated FFP was retained, as assessed by the Matrigel tube formation assay (angiogenesis was inhibited 63.7% [95% CI = 50.9% to 76.5%] after immunoprecipitation, Fig. 6
, D).
Finally, treated FFP was subjected to ion-exchange Q-Sepharose chromatography, and three fractions were obtained (the flow-through after loading at 150 mM NaCl combined with a wash at 300 mM NaCl, the eluate at 400 mM NaCl, and the wash at 1 M NaCl). Each fraction was tested for antiangiogenic activity in the Matrigel HUVEC tube formation assay. Antiangiogenic activity was detected in the fraction eluted at 400 mM NaCl (Fig 7, B) but not in the flow-through fraction (Fig. 7
, A) or the 1 M NaCl wash fraction (Fig. 7
, C). Western blot analysis of the three fractions showed that most of the angiostatin was in the flow-through but that some could also be detected in the 400 mM fraction (Fig. 7
, D). Thus, angiostatin and antiangiogenic activity clearly could be separated by affinity chromatography, immunodepletion, or ion-exchange chromatography.
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To determine whether angiostatin was responsible for the antiangiogenic effects seen in vivo with the plasma from patient 1 who was treated with rt-PA and captopril, plasma was obtained 4 hours and 8 hours into the infusion with rt-PA at 0.02 mg/kg/h, and angiostatin was immunoprecipitated with a monoclonal antibody against angiostatin. As shown by western blot analysis, high levels of angiostatin were detected in the plasma before immunoprecipitation, and angiostatin was removed by immunoprecipitation (Fig. 8, F). Angiostatin-depleted plasma retained its antiangiogenic activity, as shown by inhibition of tube formation (Fig. 8
, compare B with C and D with E).
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DISCUSSION |
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In vitro treatment of human FFP with rt-PA and captopril substantially increased the in vitro antiangiogenic activity of the plasma, as assessed by the Matrigel tube formation assay (Fig. 1) and by endothelial cell proliferation (Fig. 2
). We extended these in vitro findings to an in vivo setting by showing decreased neovascularization in the Matrigel plugs of mice treated with rt-PA and captopril (Fig. 3
). Moreover, we treated three cancer patients with a combination of captopril and low-dose rt-PA and observed the induction of an antiangiogenic effect in their plasma (Figs. 4 and 5
). Patient 1 received captopril and four doses of rt-PA, each at a successively higher concentration, and the antiangiogenic activity of her plasma increased with higher doses of rt-PA. Patients 2 and 3 received captopril and one dose of rt-PA at 0.015 mg/kg/h, which was equal to the lowest dose given to patient 1, and the antiangiogenic activity of their plasma increased slightly. These two patients were part of the first patient cohort in a phase I trial that tested rt-PA and captopril. Because the protocol did not allow intrapatient dose escalation, we could not test higher rt-PA doses in patients 2 and 3, which may explain the lower levels of antiangiogenic activity detected in the plasma of these patients compared with that of patient 1.
The fact that the plasma of patients treated with rt-PA and captopril inhibited endothelial cell proliferation and capillary tube formation suggests that a biologically relevant level of antiangiogenic activity can be induced at clinically tolerable doses of rt-PA and captopril. Moreover, the finding in the Matrigel plug assay that systemic administration of rt-PA and captopril to mice decreased neovascularization further indicates that the effects induced may be biologically important.
A novel finding that distinguishes our results from those of Gately et al. (17) and Soff (36) is that the antiangiogenic activity in FFP treated with rt-PA and captopril was independent of angiostatin. Pure angiostatin at 10 µg/mL and 50 µg/mL did not substantially inhibit tube formation (Fig. 6, A, and data not shown). LysineSepharose affinity depletion of angiostatin from FFP treated with rt-PA and captopril did not reduce the antiangiogenic activity in the treated FFP (Fig. 6
, B). Treated FFP retained antiangiogenic activity after immunoprecipitation of angiostatin (Fig. 6
, D). Ion-exchange chromatography of treated FFP demonstrated that most of the antiangiogenic activity could be separated from angiostatin (Fig. 7
).
The fact that angiostatin did not play a major role in the antiangiogenic effects of the treated plasma was unexpected. Thus, other antiangiogenic components must be generated by the treatment with rt-PA and captopril that are separable from and may be more potent than angiostatin (Figs. 6 and 7). That angiostatin did not inhibit tube formation in our assay may be related to the conditions of the assays used. In our assays (for ex vivo treatment of plasma), we resuspended the cells in full endothelial cell growth medium that is rich in multiple endothelial cell growth factors. Reports that demonstrated inhibition of tube formation by angiostatin used a less rich medium of VEGF alone, bFGF alone, or low concentrations of serum (37, 38).
What are the antiangiogenic compounds whose activity was observed in these studies? In the absence of FFP, rt-PA or captopril, alone or in combination, did not produce substantial antiangiogenic activity in vitro (Fig. 1). Consequently, the activity could arise from proteolytic cleavage of several plasma proteins by rt-PA, either by the plasminogen/plasmin pathway (i.e., angiostatin generation) or by a plasminogen-independent pathway. For example, urokinase and t-PA directly cleave other substrates present in plasma, such as fibrinogen or fibronectin (39, 40). In vivo, t-PA might be localized preferentially in the tumor stroma, as are plasminogen and fibrin, and might cleave extracellular matrix proteins to generate antiangiogenic peptides either directly or by activating a proteolytic cascade (e.g., the activation of matrix metalloproteinases by plasmin) (19, 41). Alternatively, a (mild) systemic fibrinolytic state induced by rt-PA in plasma (in vitro and in vivo) may increase the antiangiogenic activity of other plasma components, such as proteins of the coagulation cascade.
The increased antiangiogenic activity of the plasma from patient 1 persisted up to 36 hours after the infusion was stopped (Fig. 4, G). The length of this effect was unexpected. Because rt-PA is rapidly cleared from plasma (32), these antiangiogenic effects were probably not mediated directly by rt-PA but must have been mediated by newly generated molecule(s) with a relatively long half-life.
Our studies raise a number of questions. First, is the antiangiogenic effect specific for rt-PA? In the tube formation assay, the antiangiogenic activity of plasma (both FFP and plasma from cancer patients) treated with urokinase and captopril was similar to that of plasma treated with rt-PA and captopril (Merchan JR, Sukhatme VP: unpublished data). Moreover, we have observed that urokinase-treated plasma, but not rt-PA-treated plasma, induces apoptosis in cow pulmonary arterial endothelial cells and HMVEC-L cells, suggesting that urokinase may have additional substrates in the plasma whose cleavage produces these activities.
Second, what is the role of captopril in the induction of antiangiogenic activity? When we compared the numbers of microvessels in Matrigel plugs from mice treated with rt-PA and captopril with those from mice treated with rt-PA alone, captopril appeared to have made a small but probably real contribution to the antiangiogenic activity observed. In addition to stimulating generation of angiostatin, captopril may have inherent antiangiogenic effects that could be additive to or synergistic with the effects of rt-PA. Antiangiogenic properties of captopril have been reported (41), but the concentrations of captopril used for in vitro inhibition of angiogenesis were in the millimolar range. Because of its inhibitory effect on the angiotensin-converting enzyme (ACE), captopril may alter the balance between endothelial t-PA and PAI-1 (42). ACE generates angiotensin, an important regulator of endothelial PAI-1 (43) production, and degrades bradykinin, one of the most potent stimuli for synthesis and secretion of t-PA (44, 45). Captopril decreases the expression of PAI-1 in vitro and in vivo (46, 47), which might favor plasminogen activators (t-PA) over inhibitors (PAI-1) in the plasma and in the tumor stroma and, thus, enhance the series of proteolytic events that generate antiangiogenic molecules.
In the tumor microenvironment, rt-PA-generated plasmin (from plasminogen) could trigger a series of proteolytic events leading to degradation of the tumor matrix. During the initial stages of tumor angiogenesis, the formation and deposition of fibrin produce a favorable environment for new vessel formation (4649). We hypothesize that in vivo rt-PA may activate fibrin-bound plasminogen and enhance degradation of tumor stroma (and fibrin in particular), which would impede new vessel formation. This hypothesis may explain why tumors that overexpress t-PA are associated with fewer metastases in preclinical models (27) and appear to have a better prognosis (improved metastasis-free survival and overall survival) in patients with breast cancer and melanoma (28, 29).
In summary, human plasma may serve as a rich source of antiangiogenic activity. This activity can be induced at clinically achievable concentrations of rt-PA and captopril, suggesting that rt-PA and captopril should be investigated further as a therapy for cancer and other angiogenesis-dependent disorders. Studies to identify the molecules with antiangiogenic activity generated by this treatment and a clinical trial to evaluate the biologic effects of the treatment in patients with cancer are underway.
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NOTES |
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Work supported in part by seed funds from Beth Israel Deaconess Medical Center and the family of Victor J. Aresty, the Trust Family Foundation (to V. P. Sukhatme), the 2001 American Society of Clinical Oncology Young Investigator Award (to J. R. Merchan), and a Clinical Investigator Training Program Fellowship (to J. R. Merchan).
We thank D. Kiragu and K. Sampath for technical assistance and members of the Sukhatme laboratory for critically evaluating the data.
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REFERENCES |
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1 Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst 1990;82:46.[Medline]
2 Folkman J. The role of angiogenesis in tumor growth. Semin Cancer Biol 1992;3:6571.[Medline]
3 Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med 1995;333:175763.
4 Folkman J, Shing Y. Angiogenesis. J Biol Chem 1992;267:109314.
5 Kong HL, Crystal RG. Gene therapy strategies for tumor antiangiogenesis. J Natl Cancer Inst 1998;90:27386.
6 Cao Y. Therapeutic potentials of angiostatin in the treatment of cancer. Haematologica 1999;84:64350.[Medline]
7 Hanahan D, Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 1996;86:35364.[Medline]
8 OReilly MS, Holmgren L, Shing Y, Chen C, Rosenthal RA, Moses M, et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994;79:31528.[Medline]
9 OReilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 1996;2:68992.[Medline]
10 OReilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997;88:27785.[Medline]
11 Dhanabal M, Ramchandran R, Waterman MJ, Lu H, Knebelmann B, Segal M, et al. Endostatin induces endothelial cell apoptosis. J Biol Chem 1999;274:117216.
12 Dhanabal M, Volk R, Ramchandran R, Simons M, Sukhatme VP. Cloning, expression, and in vitro activity of human endostatin. Biochem Biophys Res Commun 1999;258:34552.[CrossRef][Medline]
13 Ramchandran R, Dhanabal M, Volk R, Waterman MJ, Segal M, Lu H, et al. Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin. Biochem Biophys Res Commun 1999;255:7359.[CrossRef][Medline]
14 OReilly MS, Pirie-Shepherd S, Lane WS, Folkman J. Antiangiogenic activity of the cleaved conformation of the serpin antithrombin. Science 1999;285:19268.
15 Browder T, Folkman J, Pirie-Shepherd S. The hemostatic system as a regulator of angiogenesis. J Biol Chem 2000;275:15214.
16 Wen W, Moses MA, Wiederschain D, Arbiser JL, Folkman J. The generation of endostatin is mediated by elastase. Cancer Res 1999;59:60526.
17 Gately S, Twardowski P, Stack MS, Cundiff DL, Grella D, Castellino FJ, et al. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proc Natl Acad Sci U S A 1997;94:1086872.
18 OReilly MS, Wiederschain D, Stetler-Stevenson WG, Folkman J, Moses MA. Regulation of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance. J Biol Chem 1999;274:2956871.
19 Lay AJ, Jiang XM, Kisker O, Flynn E, Underwood A, Condron R, et al. Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase. Nature 2000;408:86973.[CrossRef][Medline]
20 Mignatti P, Rifkin DB. Plasminogen activators and matrix metalloproteinases in angiogenesis. Enzyme Protein 1996;49:11737.[Medline]
21 Andreasen PA, Kjoller L, Christensen L, Duffy MJ. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 1997;72:122.[CrossRef][Medline]
22 Bajou K, Noel A, Gerard RD, Masson V, Brunner N, Holst-Hansen C, et al. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med 1998;4:9238.[Medline]
23 McMahon GA, Petitclerc E, Stefansson S, Smith E, Wong MK, Westrick RJ, et al. Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis. J Biol Chem 2001;276:339648.
24 Devy L, Blacher S, Grignet-Debrus C, Bajou K, Masson V, Gerard RD, et al. The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J 2002;16:14754.
25 Bajou K, Devy L, Masson V, Albert V, Frankenne F, Noel A, et al. Role of plasminogen activator inhibitor type 1 in tumor angiogenesis. Therapie 2001;56:46572.[Medline]
26 Pozzi A, Moberg PE, Miles LA, Wagner S, Soloway P, Gardner HA. Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization. Proc Natl Acad Sci U S A 2000;97:22027.
27 Hayashi S, Yokoyama I, Namii Y, Emi N, Uchida K, Takagi H. Inhibitory effect on the establishment of hepatic metastasis by transduction of the tissue plasminogen activator gene to murine colon cancer. Cancer Gene Ther 1999;6:3804.[Medline]
28 Ferrier CM, Suciu S, van Geloof WL, Straatman H, Eggermont AM, Koops HS, et al. High tPA-expression in primary melanoma of the limb correlates with good prognosis. Br J Cancer 2000;83:13519.[Medline]
29 Chappuis PO, Dieterich B, Sciretta V, Lohse C, Bonnefoi H, Remadi S, et al. Functional evaluation of plasmin formation in primary breast cancer. J Clin Oncol 2001;19:27318.
30 Duchin KL, McKinstry DN, Cohen AI, Migdalof BH. Pharmacokinetics of captopril in healthy subjects and in patients with cardiovascular diseases. Clin Pharmacokinet 1988;14:24159.[Medline]
31 Garabedian HD, Gold HK, Leinbach RC, Yasuda T, Johns JA, Collen D. Dose-dependent thrombolysis, pharmacokinetics and hemostatic effects of recombinant human tissue-type plasminogen activator for coronary thrombosis. Am J Cardiol 1986;58:6739.[Medline]
32 Verstraete M, Su CA, Tanswell P, Feuerer W, Collen D. Pharmacokinetics and effects on fibrinolytic and coagulation parameters of two doses of recombinant tissue-type plasminogen activator in healthy volunteers. Thromb Haemost 1986;56:15.[Medline]
33 Maeshima Y, Colorado PC, Torre A, Holthaus KA, Grunkemeyer JA, Ericksen MB, et al. Distinct antitumor properties of a type IV collagen domain derived from basement membrane. J Biol Chem 2000;275:213408.
34 Maeshima Y, Yerramalla UL, Dhanabal M, Holthaus KA, Barbashov S, Kharbanda S, et al. Extracellular matrix-derived peptide binds to alpha(v) beta(3) integrin and inhibits angiogenesis. J Biol Chem 2001;276:3195968.
35 Bootle-Wilbraham CA, Tazzyman S, Marshall JM, Lewis CE. Fibrinogen E-fragment inhibits the migration and tubule formation of human dermal microvascular endothelial cells in vitro. Cancer Res 2000;60:471924.
36 Soff GA. Angiostatin and angiostatin-related proteins. Cancer Metastasis Rev 2000;19:97107.[CrossRef][Medline]
37 Troyanovsky B, Levchenko T, Mansson G, Matvijenko O, Holmgren L. Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation. J Cell Biol 2001;152:124754.
38 Cornelius LA, Nehring LC, Harding E, Bolanowski M, Welgus HG, Kobayashi DK, et al. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol 1998;161:684552.
39 Weitz JI, Cruickshank MK, Thong B, Leslie B, Levine MN, Ginsberg J, et al. Human tissue-type plasminogen activator releases fibrinopeptides A and B from fibrinogen. J Clin Invest 1988;82:17007.[Medline]
40 Weitz JI, Leslie B. Urokinase has direct catalytic activity against fibrinogen and renders it less clottable by thrombin. J Clin Invest 1990;86:20312.[Medline]
41 Legrand C, Polette M, Tournier JM, de Bentzmann S, Huet E, Monteau M, et al. uPA/plasmin system-mediated MMP-9 activation is implicated in bronchial epithelial cell migration. Exp Cell Res 2001;264: 32636.[CrossRef][Medline]
42 Erdos EG, Skidgel RA. The angiotensin I-converting enzyme. Lab Invest 1987;56:3458.[Medline]
43 Kerins DM, Hao Q, Vaughan DE. Angiotensin induction of PAI-1 expression in endothelial cells is mediated by the hexapeptide angiotensin IV. J Clin Invest 1995;96:251520.[Medline]
44 Smith D, Gilbert M, Owen WG. Tissue plasminogen activator release in vivo in response to vasoactive agents. Blood 1985;66:8359.[Abstract]
45 Brown NJ, Nadeau JH, Vaughan DE. Selective stimulation of tissue-type plasminogen activator (t-PA) in vivo by infusion of bradykinin. Thromb Haemost 1997;77:5225.[Medline]
46 Bardos H, Juhasz A, Repassy G, Adany R. Fibrin deposition in squamous cell carcinomas of the larynx and hypopharynx. Thromb Haemost 1998;80: 76772.[Medline]
47 Bardos H, Molnar P, Csecsei G, Adany R. Fibrin deposition in primary and metastatic human brain tumours. Blood Coagul Fibrinolysis 1996;7:53648.[Medline]
48 Hall H, Baechi T, Hubbell JA. Molecular properties of fibrin-based matrices for promotion of angiogenesis in vitro. Microvasc Res 2001;62:31526.[CrossRef][Medline]
49 Simpson-Haidaris PJ, Rybarczyk B. Tumors and fibrinogen. The role of fibrinogen as an extracellular matrix protein. Ann N Y Acad Sci 2001;936:40625.
Manuscript received July 19, 2002; revised December 10, 2002; accepted December 24, 2002.
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