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Angiostatin_4.5-mediated Apoptosis of Vascular Endothelial Cells

Northwestern University Feinberg School of Medicine, Department of Medicine, Divisions of Hematology/Oncology [H. A. H., C. A. W., H. K., D. L. C., G. A. S.] and Pulmonary and Critical Care [N. C.], Chicago, Illinois 60611, and Division of Obstetrics and Gynaecology, Nottingham City Hospital, Nottingham, United Kingdom [S. U., C. A. M.]

	ABSTRACT

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Angiostatin, a proteolytic cleavage product of plasminogen, acts via a selective, yet poorly understood mechanism to potently inhibit angiogenesis (M. S. O’Reilly et al., Cell, 79: 315–328, 1994). Vascular endothelial cell proliferation assays revealed that angiostatin_4.5, a naturally occurring human isoform consisting of plasminogen kringle domains 1–4 and most of kringle domain 5 (G. A. Soff, Cancer Metastasis Rev., 19: 97–107, 2000), dose dependently reduces cell number despite the presence of a potent stimulus of proliferation. Flow cytometry using the vital dyes Hoechst 33342 and Pyronin Y revealed that 40% of both control and angiostatin_4.5-treated cells were in the proliferative phase, indicating that cell cycle progression is not impaired by exposure to angiostatin_4.5. Both bovine aortic endothelial cells and human umbilical endothelial cells were shown to undergo apoptosis in response to angiostatin_4.5. Caspases-3, -8, and -9 activation, specified by cleavage of fluorophore-conjugated specific peptide substrates, revealed a cascade of caspase activation that peaks at 36 h of angiostatin_4.5 treatment. Angiostatin_4.5 exposure induced release of cytochrome c from mitochondria in a caspase-dependent manner, but a pan-caspase inhibitor, zVAD-fmk, blocked cytochrome c release. Overall, these data indicate that human angiostatin_4.5 may function in vivo to block blood vessel formation by specifically inducing vascular endothelial cells to apoptose in a process likely involving both the intrinsic and extrinsic apoptosis pathways.

	INTRODUCTION

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Angiogenesis, the process by which new blood vessels sprout from existing vessels to vascularize tissues, is a necessary physiological process for embryonic development, wound healing, and reproductive cycles in adult females (1, 2, 3, 4, 5, 6) . Angiogenesis is also recognized as a characteristic for pathological conditions, such as psoriasis, proliferative retinopathies, and cancer growth and metastasis (3 , 7, 8, 9, 10, 11, 12) . Growth of solid tumors depends on induction of angiogenesis to provide adequate oxygen and nutrients to proliferating cells and thus avoid necrosis (13, 14, 15) . In addition, neovasculature provides a physical route for tumor metastasis (16 , 17) . Up-regulation of angiogenic stimulators, such as bFGF,³ as well as down-regulation of angiogenic inhibitors, such as thrombospondin-1, by tumor cells collectively serve to induce angiogenesis (18, 19, 20, 21) .

As initially described, angiostatin is a M_r 38–45,000 proteolytic fragment of plasminogen comprising the first three or four of the five kringle domains of plasminogen that potently inhibit angiogenesis (22, 23, 24) . It is now known that angiostatin exists as multiple isoforms differing in kringle content (22 , 23 , 25) . Antiangiogenic activity is postulated to be contingent on kringle domain composition, and various isoforms likely exhibit different levels of activity (25) . Autoproteolysis of human plasminogen, mediated by a free sulfhydryl donor, yields a M_r 52–55,000 molecule consisting of kringles 1–4 and 85% of kringle 5, which we designated as AS_4.5 and identified in human ascites and plasma (26) . To date, AS_4.5 is the only angiostatin isoform identified in human plasma. In vitro assays have revealed that isolated kringle 5 domain acts as a potent inhibitor of endothelial cell proliferation, possibly explaining why AS_4.5 inhibits endothelial cell function much more effectively than the originally described angiostatin_1–4 (kringle 1–4; Ref. 27 )_. In several previous studies, AS_4.5 was shown to exhibit potent antitumor effects in mice (26 , 28, 29, 30) ; however, the angiostatin in those studies was not referred to as AS_4.5 because the full structure was not recognized at the time.

The mechanism of angiogenic inhibition by AS_4.5, as well as other isoforms, is not fully characterized. It has been demonstrated previously in vitro that angiostatin inhibits endothelial cell migration, tube formation, and proliferation in response to growth factors (22 , 31) . As a possible explanation, two studies have concluded that angiostatin induces apoptosis in endothelial cells. In collaboration with Lucas et al. (32) , we reported previously that treatment of endothelial cells with several angiostatin isoforms, including AS_4.5, specifically led to apoptosis. However, there was no characterization of the role of AS_4.5 on cell cycle, and the nature of AS_4.5-induced apoptosis was limited. This study now characterizes the AS_4.5-induced apoptosis (32) . Likewise, Claesson-Welsh demonstrated an increase in the apoptotic index of endothelial cells exposed to angiostatin_1–3 (kringles 1–3) but found no change in the proliferative index (33) . These publications suggest that the antiangiogenic activity of angiostatin is mediated by induction of endothelial cell apoptosis. Questions as to the significance of apoptosis in mediating the physiological inhibition of angiogenesis remain, however, as both studies report low levels of apoptosis (2–20%), and it is not clear whether this effect is of sufficient magnitude to wholly explain angiostatin’s antiangiogenic action. Additionally, the studies of Claesson-Welsh et al. (25 , 33) used elastase-derived angiostatin, which lacks kringle 5, a key functional domain. Therefore, in the present study, we focused specifically on determining the mechanism of action of AS_4.5 by: (a) assessing whether its antiproliferative effect is attributable to inhibition of cell cycle progression or apoptosis; and (b) investigating the signaling pathways AS_4.5 activates to induce cytotoxicity. Through the use of cell cycle analysis and Annexin V binding, we demonstrated that AS_4.5 did not affect cell cycle progression but instead induced apoptosis. Additionally, we enlisted caspase activity assays to explore the signaling events leading to apoptotic induction.

	MATERIALS AND METHODS

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Cell Culture.
BAECs were freshly isolated from calf aortas and maintained in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (US Bio-Technologies, Parkerford, PA) and 100 µg/ml Pen-Strep (Invitrogen) and Amphotericin B (Biologos, Montgomery, IL). DMEM supplemented with 2.5% heat-inactivated calf serum (US Bio-Technologies) and 100 µg/ml Pen-Strep and Amphotericin B were used for all experimental assays testing the effects of AS_4.5 on BAECs in culture. Cells were maintained at 37°C in humidified 5% CO₂.

Angiostatin Generation.
AS_4.5 used in all assays was generated from a cell-free system in which human plasminogen was incubated with recombinant human urokinase and N-acetyl-L-cysteine (30) . The protein content of the AS_4.5 was analyzed by Western blot and measured by absorbance at 280 nm, as described previously, and tested for function (26 , 30) .

Endothelial Cell Proliferation Assay.
BAECs were plated in a 24-well tissue culture plate (Costar, Corning, NY) at a concentration of 3.3 x 10³ cells/well. The following day, the cells were grown in fresh media containing bFGF (3 ng/ml; BD Biosciences, San Jose, CA) with AS_4.5. The AS_4.5 was at 100 nM unless otherwise specified. Wells containing only media or media with bFGF served as control. Each of the conditions was performed in duplicate. After 72 h, the total numbers of cells per well were obtained using a Coulter Counter (Beckman Coulter, Miami, FL).

Cell Cycle Fractionation.
BAECs plated at a concentration of 125,000 cells/60-mm plate were grown to subconfluence. The following day, the cells were treated with fresh media containing bFGF (3 ng/ml) and AS_4.5 (100 nM) or bFGF alone. BAECs were analyzed for cell cycle status 24 h later by a method described previously by Ladd et al. (34) .

Apoptosis by Annexin V Binding Assay.
Subconfluent BAECs were grown in media supplemented with bFGF (3 ng/ml) and AS_4.5 (5.2 µg/ml) or bFGF alone. The cells were dispersed by trypsin:EDTA (Mediatech Cellgro) 48-h post-treatment and washed in fresh media with serum. The cells were further washed in PBS (Invitrogen). Early apoptotic events were then determined using the ApoAlert Annexin V kit (Clontech, Palo Alto, CA) as described by the manufacturer. Briefly, the cells were washed in 1 x binding buffer by centrifugation and then resuspended in 200 µl of 1 x binding buffer containing Annexin V (0.1 µg/ml) and PI (0.5 µg/ml). After incubation at room temperature for 15 min., the cells were analyzed by flow cytometry. Cells grown in the presence of a caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk; Enzyme Systems Products, Dublin, CA), were also tested for Annexin V binding. zVAD-fmk (50 µM) was added into the culture media in conjunction with the bFGF and AS_4.5, thus exposing the cells to the caspase inhibitor for 24 or 48 h.

Apoptosis by Ethidium Bromide and Acridine Orange Staining.
HUVECs (passage 5) were maintained on gelatinized plastic in M199 media with Earle’s salts, 20% iron-supplemented calf serum, 0.15% sodium bicarbonate, 15 units/ml heparin, 0.014 M HEPES, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10 ng/ml fibroblast growth factor and epidermal growth factor (35) . HUVECs were seeded at 20,000 cells/well (confluent) and 7,500 cells/well (subconfluent) in 96-well plates and incubated at 37°C with 5% CO₂. After 24 h, AS_4.5 was added at doses ranging from 1 ng/ml to 10 µg/ml (0.02–200 nM). As positive control, cells were also incubated with media alone and media containing 80 µg/ml etoposide. After an additional 24 h, the level of apoptosis in HUVEC cultures was determined by ethidium bromide and acridine orange staining. Briefly, the culture media were removed, and cells were washed with PBS at room temperature. Cells were incubated with ethidium bromide (10 µg/ml) and acridine orange (3 µg/ml) in PBS and examined by fluorescence microscopy. The total number of cells and number of morphologically apoptotic cells were counted for one random view in each well at x400 magnification. From these values, the percentage of apoptotic cells was determined, and the mean ± SE was calculated from four replicate samples. The significance was assessed with a Mann-Whitney U test.

Caspase Activity from Whole-cell Lysates.
Subconfluent BAECs were cultured in fresh media containing bFGF (3 ng/ml) and AS_4.5 (100 nM) or bFGF alone for 12, 24, 36, and 48 h. Cells were gently scraped from the plates and collected by centrifugation at 250 x g. Approximately 1 x 10⁶ cells were resuspended in 50 µl of cold cell lysis buffer. The caspases-3, -8, and -9 Fluorometric Assay Kits (R&D Systems, Minneapolis, MN) were used to compare the protease activity of the lysates. The lysates were diluted 1:2 in reaction buffer and incubated separately with each of the caspase-specific peptides conjugated to 7-amino-4-trifluromethyl coumarin for 1 h at 37°C. Fluorescence was measured using a microtiter plate fluorometer. To compare the caspase activity at different time points, the caspase activity was normalized to total protein concentration in the cell lysates.

Cytochrome c Release Assay.
Subconfluent BAECs were plated on 60-mm culture dishes at 20–30% confluence in media supplemented with bFGF (3 ng/ml) alone or in combination with AS_4.5 (100 nM), zVAD-fmk (50 µM), or both for 48 h. Adherent and nonadherent cells were washed with PBS and placed on a glass slide at 14,000 x g for 5 min (Cytospin 3 Cytocentrifuge, Shandon). The cells were fixed with 40% methanol (5 min, -20°C), blocked in 1% BSA (Sigma), and incubated for 2 h with 1 mg/ml anticytochrome c monoclonal antibody (BD PharMingen, San Diego, CA) at 37°C in a humidified environment. The cells were then washed in PBS containing 0.1% BSA, air dried, and incubated for 1 h with 1 mg of rhodamine-conjugated secondary antibody (Chemicon International, Temecula, CA). Subsequently, the cells were washed as before, air dried, and stained with 4',6-diamidino-2-phenylindole/1,4-diazobicyclo[2,2,2]octane for nuclear staining.

	RESULTS

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AS_4.5 Selectively and Dose Dependently Inhibits Endothelial Cell Proliferation within 1 h of Treatment.
The biological activity of AS_4.5 was tested for effect on BAEC cell number. AS_4.5 caused a concentration-dependent decrease in cell number with half-maximal effect at 24 nM and maximal effect at 100–200 nM (Fig. 1A) . After 72 h of treatment, cells treated with 100 nM AS_4.5 showed a 75–80% reduction in total cell number as compared with control cells. This effect could not be overcome by cotreatment with a variety of bFGF concentrations (3–20 ng/ml; Fig. 1B ). To determine whether the potent inhibitory effect was specific for endothelial cells, several other cell types were also treated with AS_4.5 under parallel conditions. AS_4.5 had no inhibitory effect on the growth of primary bovine smooth muscle cells, PC-3 prostate cancer cells, ECV304 bladder carcinoma, and Lewis lung carcinoma cells, confirming the selectivity of AS_4.5 for vascular endothelial cells (data not shown). Of interest, we observed that total cell number after treatment with AS_4.5 for 72 h was consistently less than the number of cells initially plated. Such data suggested a cytotoxic effect by AS_4.5.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. BAEC survival dose dependently decreases in response to AS4.5 treatment, regardless of growth factor stimulation. A, duplicate wells of subconfluent BAECs were grown in control media (low-serum media supplemented with 3 ng/ml bFGF) with or without AS4.5 in a concentration range of 10–200 nM for 72 h before counting. Percentage of cell survival (number of angiostatin-treated cells/number of control cells) represents the average of four determinations (±SE). B, experiments identical to those of A were repeated in the presence of multiple bFGF concentrations (0–20 ng/ml).

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Reduction of BAEC number in response to AS4.5 treatment occurs within 1 h. Duplicate wells of subconfluent BAECs were grown in control media (low-serum media supplemented with 3 ng/ml bFGF) with or without 100 nM AS4.5 for 1–72 h. After the shorter treatments, wells were rinsed with PBS, and control media prepared at time 0 were added. Cells were counted, and percentage of cell survival (number of angiostatin-treated cells/number of control cells) represents the average of four determinations (±SE).

Hence, these data indicate that the reduction noted previously in BAEC number on AS_4.5 treatment is not caused by a block on proliferation and further supports the hypothesis that the potent inhibitory effect of AS_4.5 on BAEC may be caused by a cytotoxic effect.

Apoptosis Induction by AS_4.5.
Based on previous studies, apoptosis was hypothesized to account for the cytotoxic effect of AS_4.5 alluded to above (32 , 33) . To elucidate whether AS_4.5 induces endothelial cell apoptosis, annexin V binding was done in conjunction with PI staining to detect apoptosis and distinguish early stage apoptotic cells, Annexin V positive, from late stage apoptotic and necrotic cells, dual Annexin V and PI positive. Forty-eight h of AS_4.5 treatment caused 50% of the endothelial cells to bind Annexin V, marking the early stages of apoptosis, whereas only 2% of control cells were apoptotic (Fig. 3) . This is a markedly greater effect than had been observed previously with angiostatin_1–4 (kringles 1–4; Refs. 32 and 33 ).

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. AS4.5 induces BAEC to apoptose. FITC-conjugated Annexin V was used to detect cells in the early stages of apoptosis by flow cytometry. Cells cultured in the absence or presence of the indicated drugs for 48 h were then incubated with Annexin V and PI and subjected to flow cytometric analysis. The bottom right quadrant (Annexin V+) of each dot plot represents cells in the early stages of apoptosis, whereas the top right quadrant (Annexin V+, PI+) represents cells in late stage apoptosis or necrosis. A, control (low-serum media supplemented with 3 ng/ml bFGF); B, 100 nM AS4.5; C, 50 µM zVAD control; D, 100 nM AS4.5 and 50 µM zVAD-fmk. Annexin V binding analysis was preformed more than three times, with comparable results.

Using acridine orange and ethidium bromide staining, early passage human umbilical endothelial cells were also shown to undergo apoptosis in response to AS_4.5 (Fig. 4) . After 24 h of AS_4.5 treatment, 40% of the cells were apoptotic, showing condensed chromatin uniformly stained by acridine orange, in both confluent and subconfluent conditions. Maximal effect was observed at 20–200 nM. In negative control cells, apoptosis was not detected (<0.5%). Thus, both human and bovine endothelial cells were shown to undergo apoptosis in response to AS_4.5.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. AS4.5 dose dependently increases HUVEC apoptosis. Confluent (20,000/well) or subconfluent (7,500/well) cultures of HUVECs were exposed to AS4.5 at a range of concentrations for 24 h. In parallel, HUVECs were treated with 80 µg/ml etoposide (positive control) or HUVEC media (negative control). After staining with ethidium bromide (10 µg/ml) and acridine orange (3 µg/ml) in PBS, the number of apoptotic cells as a proportion of the total cells was measured using fluorescence. Data are mean ± SE of the percentage of apoptotic cells from four replicates.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Activation kinetics of caspases-3, -8, and -9 in response to AS4.5. BAECs were treated with 100 nM AS4.5 for 12, 24, 36, or 48 h. Lysates prepared from each time point were assessed for specific activation of caspases-8, -9, and -3. The reaction was initiated by adding the appropriate fluorogenic caspase substrate to 50 µl of lysate. Product formation was measured after 1 h of incubation at 37°C. Bradford protein assays were performed on each lysate to normalize relative fluorescence unit values. Data are shown as the fold increase of caspases-8, -9, and -3 activity in angiostatin-treated cells as compared with untreated cells with time. Caspases-8 and -9 assays were performed in two separate experiments with comparable results; caspase-3 activity as measured separately by colorimetric assay also showed comparable results.

Activation of caspase-3 is a relatively late event in apoptosis. Two distinct pathways are capable of initiating caspase-3 activation. An intrinsic or mitochondrial apoptotic pathway generates active caspase-9, which then directly activates caspase-3. Additionally, an extrinsic, or surface receptor apoptotic pathway, generates active caspase-8, which can then activate caspase-3 directly as well as indirectly via activation of the intrinsic pathway (36) . Elucidation of the upstream activator of caspase-3 may specify the pathway of apoptotic initiation. Therefore, activation of caspases-8 and -9 was investigated using fluorogenic peptide assays. Although control BAECs showed a steady, low level of caspases-8 and -9 activity, AS_4.5 treatment for 12, 24, 36, and 48 h induced sharp peaks in both activities. Caspase-8 showed a 2-fold activity increase at 12 and 24 h and peaked with a 6-fold increase in activity at 36 h (Fig. 5) . Similarly, caspase-9 activity was also modestly higher in BAECs exposed to AS_4.5 for 12 and 24 h but spiked to 17-fold induction at 36 h. These data implicate a synchronized apoptotic signaling cascade of caspases-8, -9, and -3 in response to AS_4.5.

AS_4.5-induced Apoptosis Is Characterized by Caspase-dependent Release of Cytochrome c.
Active caspase-9 exists as a holoenzyme, or apoptosome, consisting of caspase-9, APAF-1, and cytochrome c. Cytochrome c is constitutively housed within the mitochondrial intermembrane compartment and must be released in order for activation of caspase-9 to occur (36) . Release of cytochrome c, as measured by a specific antibody, was detected in response to 100 nM AS_4.5 exposure for 48 h but was blocked by 50 µM zVAD-fmk, indicating that its release is a caspase-dependent process (Fig. 6) .

View larger version (14K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. AS4.5 treatment initiates caspase-dependent release of cytochrome c. Subconfluent BAECs were grown with AS4.5 (100 nM), zVAD-fmk (50 µM), or both for 48 h, placed on a glass slide, fixed with methanol, and blocked in 1% BSA. Cytochrome c release was measured by an anticytochrome c monoclonal antibody, followed by a rhodamine-conjugated secondary antibody, and quantified by fluorescence.

	DISCUSSION

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Our findings demonstrate and characterize AS_4.5-induced apoptosis in endothelial cells, both human and bovine. We have shown that AS_4.5 dose dependently reduced endothelial cell number in proliferation assays, despite the presence of increasing amounts of bFGF, a potent stimulus of proliferation, indicating that angiostatin is not competitive with bFGF. Flow cytometry with the vital dyes Hoechst 33342 and Pyronin Y indicated that cell cycle progression is not impaired by exposure to AS_4.5, suggesting cytotoxicity and negating classic growth inhibition as the mechanism of action. Annexin V staining verified the cytotoxic effect of AS_4.5 and identified apoptosis as the death mechanism. Fluorogenic peptide substrates specific for the primary caspases-3, -8, and -9 revealed a cascade of caspase activation that peaks with 36 h of AS_4.5 treatment. Finally, caspase-dependent release of cytochrome c from mitochondria on AS_4.5 treatment was demonstrated by staining. Overall, we demonstrated, for the first time, that AS_4.5 treatment selectively induces endothelial cells to undergo a cell death that is biochemically identical to apoptosis.

Moreover, we have shown that AS_4.5 is a highly potent isoform. With a maximal effect of 75–80% reduction in cell number and half-maximal dose of only 24 nM in BAEC proliferation assays, AS_4.5 appears to be more potent than smaller isoforms lacking kringle 5 domain. Experiments in our lab find angiostatin_K1–3 to possess 60% the potency of AS_4.5 in BAEC proliferation assays (data not shown). The magnitude of the apoptotic effect of AS_4.5 (40% in HUVEC and 50% in bovine endothelial cells) is greater than the 2–20% that had been reported previously by Lucas et al. (32) . This may reflect the greater potency of AS_4.5 compared with angiostatin_1–4 (23 , 27 , 38) or the more sensitive apoptosis assays currently available. Unfortunately, limited availability of the various isoforms prevents extensive direct comparison at this time.

We also reveal that AS_4.5 has no antiproliferative effect. Endothelial cell number drops on treatment with AS_4.5, but surviving cells continue to proceed to S phase of the cell cycle as indicated by accumulating DNA and RNA content. These data therefore indicate that the antiproliferative effect is not attributable to cell cycle block and thus further support the hypothesis that the potent inhibitory effect of AS_4.5 on BAEC, and likely angiogenesis, may be attributable to induction of apoptosis.

Additionally, this study demonstrates for the first time that a short exposure (1 h) to AS_4.5 is sufficient to induce a potent antiangiogenic effect despite extensive washout of the drug, implying that an irreversible process, i.e., apoptosis, is initiated. Such knowledge is beneficial to the therapeutic application of AS_4.5, because continuous exposure to angiostatin may not be necessary for an inhibition of angiogenesis. Along the same lines, low concentrations (24–100 nM) are found to induce an effect, again indicating clinical potential for use of AS_4.5 as an anticancer agent.

Although two papers have suggested previously that angiostatin induces apoptosis in endothelial cells, this study is the first to characterize the biochemical hallmarks of apoptosis. Here, we demonstrate activation of a caspase cascade in response to AS_4.5; activation of caspases-8, -9, and -3 appeared with 12 h of AS_4.5 treatment and peaked with 36 h of treatment. Furthermore, the pan-caspase inhibitor zVAD-fmk completely protected endothelial cells from apoptosis, presumably by blocking the caspase activation cascade. Activation of caspase-8 implicates involvement of the death receptor pathway for initiation of apoptosis, but release of cytochrome c and subsequent activation of caspase-9 indicate that the mitochondrial pathway is involved as well. Importantly, blockade of AS_4.5-mediated cytochrome c release by zVAD-fmk treatment indicates that this release is a caspase-dependent mechanism. Active caspase-8 has been demonstrated to induce activation of caspase-9 via cleavage of Bid to yield tBid, which can induce the release of cytochrome c (39 , 40) . Caspase-8 activity is effectively blocked by zVAD-fmk. Overall, these data implicate the surface receptor pathway and caspase-8 activation as the initiators of apoptosis. As yet, the relative contributions of the surface and mitochondrial pathways remain to be clarified; additional work will address these issues.

Additionally, the factor(s) on the cell surface by which AS_4.5 initiates the surface pathway for apoptosis remains to be elucidated. Moser et al. (41 , 42) have proposed that a membrane ATP synthase on the surface of endothelial cells is a receptor for angiostatin_K1–3. They hypothesized that angiostatin-mediated inhibition of the ATP synthase may be responsible for the antiangiogenic effect, either by hindering production of extracellular ATP or by inhibiting the proton pump function of the ATP synthase (43) . It is not clear if ATP synthase represents a target for the human AS_4.5 system, because the studies of Moser used a different angiostatin isoform, lacking the kringle 5 domain, and significantly more angiostatin_K1–3 (i.e., 0.5–1 µM) was required for function than we observe for AS_4.5.

In addition, Troyanovsky et al. (44) have shown that a novel endothelial cell protein, angiomotin, has a role in mediating migration and that binding of angiostatin_K1–4 to angiomotin inhibits endothelial cell migration. It is not known if angiomotin has a role in AS_4.5-mediated apoptosis, because their studies also used a different angiostatin isoform, lacking the kringle 5 domain, and they reported no studies on the role of angiomotin on cell proliferation or apoptosis. In unpublished studies, we have found that induced expression of angiomotin, by expression vector transfection, failed to convey AS_4.5 responsiveness to AS_4.5-resistant endothelial cell lines, including EAHy926.

The conclusive demonstration of endothelial cell apoptosis by AS_4.5 raises some intriguing questions about the essential process of angiogenesis inhibition. The term "angiostatin" was coined to reflect the presumed ability of the protein to inhibit new blood vessel growth, i.e., angiogenesis. It was believed by our group and others that angiostatin acted by slowing the growth of new vessels. If AS_4.5-induced apoptosis of vascular endothelial cells, observed in vitro, is also the mechanism of action of AS_4.5 in vivo, then AS_4.5 is not actually angiostatic but angiocidal. In that case, the question of which vascular endothelial cell populations are affected by AS_4.5, (i.e., proliferative versus quiescent, tumor-associated versus normal) must be addressed for AS_4.5 to be translated to a practical therapy.

	FOOTNOTES

 
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.

¹ Supported by grants from the National Cancer Institute (P50 CA90386), P50 CA89018-02), and (R21 CA89886-01), and a grant-in-aid from the Hairy Cell Leukemia Research Foundation.

² To whom requests for reprints should be addressed, at Northwestern University Feinberg School of Medicine, Department of Medicine, Divisions of Hematology/Oncology, Chicago, IL 60611. Phone: (312) 695-4442; Fax: (312) 695-6189; E-mail: g-soff{at}northwestern.edu

³ The abbreviations used are: bFGF, basic fibroblastic growth factor; BAEC, bovine aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; PI, propidium iodide; AS_4.5, angiostatin_4.5.

Received 12/27/02. Accepted 5/ 7/03.

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