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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Angiogenesis Inhibitors Target the Endothelial Cell Cytoskeleton through Altered Regulation of Heat Shock Protein 27 and Cofilin

Susan M. Keezer, Susan E. Ivie, Henry C. Krutzsch, Anita Tandle, Steven K. Libutti and David D. Roberts
DOI:  Published October 2003

Abstract

Inhibition of angiogenesis has emerged as a key focus for the treatment of cancer, necessitating a better understanding of the downstream molecular targets of angiogenesis inhibitors. Endostatin, thrombospondin-1, fumagillin, and its synthetic derivative, TNP-470, are potent inhibitors of endothelial cell proliferation and migration in culture and of angiogenesis in vivo. To identify targets that mediate the effects of these inhibitors, we compared two-dimensional gel electrophoresis patterns from lysates of treated and untreated human endothelial cells. Among the proteins identified were cofilin and hsp27, two proteins involved in actin dynamics. Western blotting and immunofluorescence experiments confirmed that the phosphorylation states and subcellular localization of these two proteins were affected by all of the inhibitors tested and that treated cells had a more extensive network of actin stress fibers and more numerous focal adhesion plaques compared with untreated cells. Endothelial monocyte activating polypeptide II, another angiogenesis inhibitor, elicited the same response in the actin cytoskeleton and focal adhesions of endothelial cells. This more adherent phenotype may explain the shared ability of these inhibitors to block endothelial migratory signals. Starting with a proteomics approach, we have identified common effector molecules used by a panel of angiogenesis inhibitors that perturb the cytoskeleton to prevent endothelial migration.

INTRODUCTION

The process of angiogenesis, where new capillaries are engendered from existing blood vessels, occurs under normal physiological conditions and in several pathological states. In order for angiogenesis to occur, endothelial cells, stimulated by a high ratio of positive to negative angiogenic factors, invade and migrate through the extracellular matrix, proliferate, and finally form tubular structures at the site to be vascularized (1, 2, 3) . Tumor angiogenesis has become of particular interest to cancer researchers. Angiogenesis from blood vessels adjacent to the growing tumor cells is required for an in situ carcinoma to become malignant and metastasize (4) . Inhibition of angiogenesis using drugs that act on endothelial cells is an attractive way of treating cancer patients, because it would transcend the problems of tumor heterogeneity and drug resistance commonly encountered when the cancer cells themselves are targeted for treatment (5) . Several inhibitors of angiogenesis have been tested in clinical trials with limited success (6) . Although the effects of these inhibitors on endothelial cells are clearly detrimental to the process of angiogenesis, our understanding of the means by which this inhibition is accomplished is inadequate for us to predict how they should be used therapeutically (7, 8, 9, 10) .

To improve the effectiveness of angiogenesis inhibition, it is important that we understand the mechanisms of action used by each inhibitor. For this purpose, others have identified targets of angiogenesis signaling by searching for alterations in gene expression in response to proangiogenic factors or in tumor versus normal endothelial tissue (11, 12, 13) . We propose that, although known angiogenesis inhibitors use distinct receptors to elicit the same effects on endothelial cells, they share downstream target molecules required for their antiangiogenic functions. Here, we used a proteomic approach to identify such common targets of a panel of angiogenesis inhibitors.

The inhibitors used in this study are the fungal product fumagillin, its synthetic analogue, TNP-470, thrombospondin-1, endostatin, and EMAP 3 II. Fumagillin and TNP-470 inhibit endothelial cell proliferation and block angiogenesis in vitro and in vivo (14 , 15) . 4 These two molecules interact with methionine aminopeptidase-2, although it is unclear if this binding is necessary or sufficient for their antiangiogenic effects (16 , 17) . Thrombospondin-1, a natural modulator of angiogenesis, exhibits antiangiogenic activity in vivo and in vitro (18, 19, 20, 21, 22) . Through different domains, thrombospondin-1 can bind to several endothelial cell receptors. Of these, CD47, heparan sulfate proteoglycans, and CD36 mediate antiangiogenic responses (reviewed in Ref. 23 ). Endostatin, a fragment of collagen XVIII, is also a natural inhibitor of angiogenesis that diminishes tumor growth in mouse models and inhibits endothelial cell migration in vitro (24, 25, 26) . Endostatin’s receptors include integrins and glypican (27 , 28) , and it modulates VEGF and Wnt signaling pathways as well as matrix metalloprotease-2 activation (29, 30, 31, 32) . EMAP II is an antiangiogenic cytokine shown to cause endothelial cell apoptosis and affect monocyte and macrophage chemotaxis (33, 34, 35, 36) .

Using two-dimensional gel electrophoresis to compare proteomic responses of human endothelial cells with some of these inhibitors, we discovered consistent changes in abundance and/or post-translational modification of several cytoskeletal proteins, including hsp-27 and cofilin. Both of these proteins are involved in actin dynamics and are regulated through differential phosphorylation and subcellular localization. Hsp-27, in addition to its function in protein folding assistance during heat shock, possesses actin-capping activity that regulates actin polymerization (37) . Phosphorylation of hsp27 is associated with changes in the actin cytoskeleton in VEGF-stimulated endothelial cell motility (38) . Cofilin is an actin depolymerizing protein involved in cellular processes that require actin turnover, such as motility and cytokinesis (39, 40, 41, 42, 43, 44, 45) . Cofilin was also identified previously as an up-regulated gene by microarray in ovarian adenocarcenomas versus normal ovary in a screen that also identified some known angiogenic molecules (46) . Here we show that cofilin and hsp27 phosphorylation and subcellular localization are altered in response to angiogenesis inhibitors. Under the same conditions, actin stress fiber and focal adhesion densities are increased, indicating that angiogenesis inhibitors induce endothelial cells to assume an adhesive state that is not conducive to motility. These data reveal a possible common mechanism used by a panel of angiogenesis inhibitors to inhibit endothelial cell migration.

MATERIALS AND METHODS

Cell Culture.

HUVECs were obtained from Biowhittaker and maintained in M199 E (Biosource International) supplemented with 20% fetal bovine serum, 2 mm glutamine, penicillin and streptomycin. HDMVE cells were obtained from Biowhittaker and maintained in EBM-2 supplemented with EGM-2 MV singlequots (Biowhittaker). All endothelial cells were used in passages four through seven. Jurkat T cells were maintained in RPMI 1640 (Biosource International) supplemented with 10% FBS, 2 mm glutamine, penicillin, and streptomycin. All cells were grown at 37°C and 5% CO2.

Inhibitors.

Recombinant human endostatin was obtained from EntreMed (Rockville, MD); dissolved in 17 mm citric acid, 66 mm sodium phosphate, 59 mm NaCl (pH 6.2) at 1 mg/ml; and used at 1 μg/ml. Fumagillin was obtained from Sigma Biochemicals, dissolved in DMSO at 10 mm, and used at 0.1 μm. TNP-470 was obtained from the National Cancer Institute/NIH Developmental Therapeutics Program and used at 0.2 μm. Thrombospondin-1 was prepared from human platelets obtained from the NIH blood bank (47) and used at 20 μg/ml. EMAP-II preparation: DH5a cells transformed with pET-20b plasmid, expressing mature EMAP-II as a histidine-tagged protein, were obtained from Paul Schimmel at The Scripps Research Institute (La Jolla, CA). The bacterial cultures were induced with isopropyl-β-d-thiogalactoside (Sigma) and lysed in lysis buffer [50 mm Tris-HCL (pH 7.5), 0.15 m NaCl, and 10% sucrose] containing 200 μg/ml lysozyme. Triton-X-100 was added at a final concentration of 1%, and the lysate was incubated at 4°C for 30 min. The samples were centrifuged at 15,000 rpm for 30 min. The supernatants were incubated with Ni-NTA Superflow affinity resin (Qiagen; 1 ml of bed volume per 100 ml supernatant) for 1 h at 4°C. The fusion protein was eluted from the affinity column using increasing concentrations (10, 50, 100, and 200 mm) of Imidazole (Fisher Biotech). The fractions were screened for the presence of biologically active EMAP-II using tissue factor assay (35) . EMAP-II was eluted at 100 mm Imidazole. Protein concentrations were determined using the BCA protein assay kit (Pierce), and the purity of the protein was confirmed by SDS-PAGE.

Two-dimensional Gel Electrophoresis.

Cellular extracts were made by lysing cells in 0.5% SDS at 100°C. The lysate was then reduced and alkylated by adding 5 mm tributylphosphine, 2.5% acrylamide, and 20 mm sodium bicarbonate. Samples were dialyzed overnight against 2L 50 mm ammonium bicarbonate at 4°C, lyophilized to dryness, and resuspended in 8 m urea. Protein concentration was determined using a BCA assay kit (Pierce). Rehydration buffer (7 m urea, 2 m deionized thiourea, 4% CHAPS, and 2% IPG buffer; Amersham/Pharmacia), 2 mm tributylphosphine) was added to 500 μg of protein per sample, and the sample was focused on a pH 3–10 nonlinear IPG strip on the IPGphore focusing system (Amersham/Pharmacia) using a program which allowed rehydration for 12 h, then focusing at 500 V for 1 h, 1000 V for 1 h, and 8000 V for 10.5 h. The strips were frozen in glass tubes at −70°C overnight. The strips were thawed and equilibrated in 1 × NuPage equilibration buffer (Invitrogen) for 30 min at room temperature. Proteins were resolved in the second dimension using a 10% NuPage BT gel (Invitrogen). Gels were stained using a colloidal blue staining kit (Invitrogen) according to the manufacturer’s instructions. Differentially localized protein spots were excised and subjected to liquid chromatography/mass spectrometry as described previously (48) .

Antibodies and Immunofluorescence Reagents.

Antipaxillin monoclonal antibody was obtained from BD Transduction Laboratories and used at a dilution of 1:1000. Anticofilin polyclonal antibody was obtained from Cytoskeleton (Denver, CO) and used at a dilution of 1:1000 for Western blot and 1:100 for immunofluorescence. Antiphospho-cofilin/ADF polyclonal antibody was a generous gift from J. Bamburg (Colorado State University, Fort Collins, CO; Ref. 49 ) and was used at a dilution of 1:5000 for Western blot and 1:200 for immunofluorescence. Anti-hsp27, anti-hsp27/phospho-Ser15, anti-hsp27 phospho-Ser78, and rabbit antisheep HRP were obtained from Upstate Biotechnology and used at dilutions of 1:500, 1:100, 1:100, and 1:2000, respectively. Goat antirabbit HRP was obtained from Sigma Biochemicals and used at a dilution of 1:10,000. Bodipy FL goat antirabbit, Bodipy TR-X phallacidin, and Alexa Fluor 488 donkey antimouse were obtained from Molecular Probes and used at dilutions of 1:1000, 1:20, and 1:500, respectively.

Immunofluorescence.

HUVE or HDMVE cells were plated onto eight-well glass chamber slides (Lab-Tek) at 10,000 cells/well or on glass coverslips in 24-well plates (Nunc) at 20,000 cells/well in complete medium. Immunofluorescence experiments were carried out 48 h after plating (cells were ∼50% confluent) after inhibitor treatment as indicated. Cells were fixed in 3.7% formaldehyde (made freshly from paraformaldehyde and adjusted to pH 10) for 10 min at room temperature. Cells were washed twice for 5 min with DPBS and blocked with 1%BSA/DPBS for 20 min at room temperature. Cells were then permeabilized in blocking buffer plus 0.2% Triton X-100 for 5 min at room temperature followed by antibody treatment in permeabilization buffer. Primary antibodies were incubated with cells for 2–3 h at room temperature, whereas secondary antibodies and phallacidin were incubated with cells for 1 h at room temperature. Cells were washed three times in DPBS after each antibody treatment and allowed to dry before examination.

Western Blotting.

Cellular extracts (105 cells/sample) were resolved by SDS-PAGE, and gels were transferred to Immobilon-P membrane (Millipore) via semidry transfer apparatus (Bio-Rad) according to the manufacturers’ instructions. Membranes were blocked for 1 h at room temperature in PBS + 0.05% Tween 20 + 5% dry nonfat milk. Primary antibody incubations were carried out for 2 h in blocking buffer, except for antiphospho-Ser15 hsp27 and antiphospho-Ser78 hsp27, which were incubated overnight at 4° in wash buffer (PBS + 0.05% Tween 20). Secondary antibody incubations were carried out for 1 h at room temperature in blocking buffer. Blots were incubated in wash buffer for 10 min after blocking, 3 × 10 min after primary antibody, and 5 × 10 min after HRP-conjugated secondary antibody. Reacting bands were detected using enhanced chemiluminescence (Pierce Supersignal West Pico).

Cellular Protein Extraction.

For two-demensional gel electrophoresis, endothelial cells were grown to ∼70% confluence in four 175 cm2 culture flasks (Nunc)/sample. Cells were treated with angiogenesis inhibitors for 24 h and removed from the flasks using 2.5 mm EDTA in PBS. Cells were lysed by boiling for 5 min in 3 ml of 0.5% SDS and allowed to cool for 5 min at room temperature. The lysate was reduced and alkylated by adding 5 mm tributylphosphine, 2.5% w/v acrylamide, and 20 mm sodium bicarbonate and incubating for 30 min at room temperature. Samples were dialyzed against 50 mm ammonium bicarbonate and 0.05% SDS overnight at 4°C, lyophilized to dryness, and suspended in 1 ml of 8 m urea. Protein concentration was determined using Pierce BCA assay. For Western blot, whole cell extracts were prepared by lysing 105 cells in 50 μl of 95°C 1 × SDS sample buffer [80 mm Tris-HCl (pH 6.8), 2% SDS, 15% glycerol, 1% β-mercaptoethanol, and 0.01% bromphenol blue] and heating the suspension at 95°C for 10 min. These extracts were prepared freshly for each experiment. To prepare subcellular extracts, cells were grown to ∼70% confluence on 150-mm tissue culture plates (Falcon), treated for 24 h as indicated, and scraped off of the plates in PBS. Cells from four plates were washed twice in 50 ml of 10 mm HEPES (pH 7.5), 10 mm KCl, and 1.5 mm MgCl2. Cells were pelleted, suspended in five volumes of the above buffer plus protease and phosphatase inhibitors (1 μg/ml leupeptin, 1 μg/ml pepstatin, 1 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 0.1 mm Na3VO4, and 50 mm NaF), and allowed to swell on ice for 10 min. The cell suspension was dounce homogenized 15 strokes on ice and centrifuged at 4°C for 10 min at 3000 rpm in a microcentrifuge. The resulting supernatant is the cytoplasmic extract. The pellet was then suspended in 500 μl of 2 mm HEPES (pH 7.4), 0.2 mm EDTA, 20% glycerol, 0.3 m (NH4)2SO4, plus protease and phosphatase inhibitors at concentrations listed above, and incubated at 4°C for 30 min rocking to extract soluble proteins. This mixture was then centrifuged at 100,000 × g at 4°C for 45 min. The resulting supernatant is the soluble nuclear extract. The pellet was suspended in 500 μl of 2 mm HEPES, 0.2 mm EDTA, 20% glycerol, plus protease and phosphatase inhibitors, and sonicated at 4°C on ice for 1 min of 3-s pulses. This is the insoluble extract. All samples were suspended in an equal volume of 2 × SDS sample buffer and boiled for 5 min before electrophoresis.

RESULTS

Endothelial Cell Cytoskeletal Proteins Migrate Differentially on Two-dimensional Gels in Response to Angiogenesis Inhibitors.

To detect changes in cellular proteins induced by angiogenesis inhibitors, we treated HUVE or HDMVE cells for 24 h with fumagillin, TNP-470, or endostatin. Whole cell extracts from treated and untreated cells were subjected to two-dimensional electrophoresis. Differentially localized protein spots were then excised, and the proteins were identified from mass spectra of tryptic peptides using Sequest and Mascot software. Among the proteins identified in this screen, several are known cytoskeleton-associated proteins (Table 1) ⇓ . As indicated, some of the proteins identified showed changes in isoelectric point or abundance that were dependent on the inhibitor used. Some of these proteins may be the targets of convergent pathways that mediate the antiangiogenic effects of each inhibitor. Alternatively, the divergent responses to the inhibitors used here may be related to processes other than inhibition of angiogenesis. We therefore chose to focus on two proteins that were consistently affected by the inhibitors in both HUVE and HDMVE cells: the actin severing protein cofilin and the actin capping protein hsp27 (Fig. 1) ⇓ . Because we did not choose specific proteins for sequencing but, rather, sequenced unknown spots whose intensity consistently changed because of treatment with angiogenesis inhibitors, the disappearance of the indicated hsp27 spots in response to endostatin or TNP-470 may be the result of either a decrease in total protein or a change in isoelectric point. In the case of cofilin, we identified a basic spot that was more abundant in untreated cells and acidic spots that were increased in inhibitor-treated cells indicating the likelihood of a post-translational modification.

Fig. 1.
Fig. 1.

The isoelectric points of cofilin and hsp27 on two-dimensional gels change in response to angiogenesis inhibitors. HUVE (A and B) or HDMVE (C and D) cell extracts were subjected to two-dimensional gel electrophoresis followed by colloidal blue staining (Invitrogen). The indicated spots were identified by liquid chromatography/mass spectrometry and searching for known human protein sequences using Mascot. Shown are the results from cells treated for 24 h with 0.1 μm fumagillin (A and C), 1 μg/ml endostatin (B), or 0.2 μm TNP-470 (D) before cell lysis. Similar results were obtained when HUVECs were treated with 0.2 μm TNP-470.

View this table:
Table 1

Observed changes in endothelial cytoskeletal proteins on two-dimensional gels

Whole HUVE or HDMVE cell protein extracts were resolved on two-dimensional gels after treatment with angiogenesis inhibitors as described in “Materials and Methods.” Protein spots whose intensity differed in treated versus untreated samples were excised identified using liquid chromatography/mass spectrometry/mass spectrometry. Known cytoskeletal proteins whose position changed in response to the inhibitors and observed changes are summarized.

Hsp27 Phosphorylation and Subcellular Localization Are Altered by Angiogenesis Inhibitors.

Hsp27 is involved in modulation of the actin cytoskeleton through its actin capping activity. This protein has several phosphorylation sites, including Ser15 and Ser78. Phosphorylation at these residues alters the subcellular localization and activity of hsp27, although the precise effects of phosphorylation are still controversial (37 , 50) . We were unable to detect any changes in subcellular localization of this protein by immunofluorescence (data not shown). Changes were, however, evident on Western blotting of subcellular protein extracts after angiogenesis inhibitor treatment (Fig. 2) ⇓ . We treated HUVECs with TNP-470 for 24 h and resolved subcellular protein fractions by SDS-PAGE. Because phosphorylation of hsp27 has been shown previously to cause dissociation of large oligomers of the protein (51) , which may be less extractable than the monomeric protein or smaller oligomers, we thought it important to fractionate the cells and compare nuclear, cytosolic, and insoluble protein extracts. Western blots were performed using polyclonal antibodies recognizing total hsp27, phospho-Ser15 hsp27, or phospho-Ser78 hsp27. The resulting image shows that phosphorylation at both Ser15 and Ser78 is increased with TNP-470 (Fig. 2A) ⇓ . Translocation of both total and phosphorylated hsp27 to the nuclear fraction is also apparent, although cytoskeletal contamination of the nuclear extracts cannot be ruled out. These data indicate that TNP-470 causes altered regulation of hsp27. The phosphorylated forms of hsp27 were increased in response to 0.1 μm fumagillin, 1 μg/ml endostatin, and 20 μg/ml thrombospondin-1 (Fig. 2B) ⇓ , identifying hsp27 as a common target of these angiogenesis inhibitors.

Fig. 2.
Fig. 2.

Angiogenesis inhibitors cause altered subcellular localization and phosphorylation of hsp27. In A, HUVECs were treated for 24 h with 0.2 μm TNP-470. Subcellular fractionation was performed by hypotonic swelling and mechanical disruption of cells (C, cytosolic extract), followed by salt extraction [0.3 m (NH4)2SO4] of nuclei (N, nuclear extract) as described in “Materials and Methods.” The remaining material after high-speed centrifugation of the salt-extracted nuclei was designated as the insoluble extract (I). In B, HUVECs were treated with 0.1 μm fumagillin (F), 0.2 μm TNP-470 (T), 0.2 μg/ml endostatin (E), 20 μg/ml thrombospondin-1 (TSP-1), or left untreated (UT) for 24 h. A and B, cell extracts were resolved using 12% SDS-PAGE, and Western blots were performed using rabbit anti-hsp27, sheep anti-hsp27/phospho-Ser15, or sheep anti-hsp27/phospho-Ser78. Blots using different antibodies were performed in parallel, and protein loading was determined to be equal by Ponceau S (Sigma) staining (data not shown).

Fumagillin, TNP-470, Endostatin, EMAP-II, and Thrombospondin-1 Increase the Ratio of Phospho-cofilin to Total Cofilin.

Because cofilin activity is regulated by phosphorylation (52) , we hypothesized that the changes on the two-dimensional gels were caused by differential phosphorylation. Whole cell protein extracts from HUVECs that were untreated or treated with the indicated angiogenesis inhibitors were resolved on one-dimensional SDS-PAGE. Western blots were then performed using either polyclonal anticofilin, which recognizes all cofilin, or polyclonal antiphospho-cofilin/ADF, which recognizes phosphorylated cofilin and ADF, an actin depolymerizing protein paralogous to cofilin. ADF has a higher rate of mobility than cofilin on SDS-PAGE and cannot be seen by Western blot in soluble extracts from these cells (data not shown), indicating that cofilin is the predominant actin-depolymerizing protein under the conditions used. The ratio of phosphorylated cofilin:total cofilin was increased after treatment of HUVECs with all angiogenesis inhibitors tested (Fig. 3) ⇓ . In Jurkat T cells, however, the constitutively higher ratio of phosphorylated to total cofilin was unaltered by the inhibitors (Fig. 3) ⇓ , providing evidence for endothelial cell specificity. Cofilin’s activity in stimulating actin depolymerization is inhibited by phosphorylation at serine 3 (52) . Therefore, these data indicate that angiogenesis inhibitors cause inactivation of cofilin.

Fig. 3.
Fig. 3.

Angiogenesis inhibitors cause increased phosphorylation of cofilin in endothelial cells. In A, HUVE or Jurkat T cells were treated for 24 h with 0.1 μm fumagillin (F), 0.2 μm TNP-470 (T), 0.2 μg/ml endostatin (E), 20 μg/ml thrombospondin-1 (TSP-1), or untreated (UT). Cellular proteins (whole cell extracts) were resolved on a 12% SDS-PAGE, and Western blotting was performed using either rabbit polyclonal anticofilin or rabbit polyclonal antiphospho-cofilin/ADF. Blots using different antibodies were performed in parallel, and protein loading was determined to be equal by Ponceau S (Sigma) staining (data not shown). B, the ratio of phosphorylated to total protein was calculated using ImageQuant. These results are the average of two or three independent experiments for Jurkat and HUVECs, respectively. SE = 0.075–0.51

Subcellular Localization of Cofilin Is Altered in Response to Angiogenesis Inhibitors.

Active cofilin can be localized to the plasma membrane (53) . To test whether phosphorylation of cofilin perturbs this localization in endothelial cells, we treated HUVECs with 0.1 μm fumagillin for 24 h and subjected them to immunofluorescence using anticofilin or antiphospho-cofilin/ADF antibodies. The resulting images (Fig. 4, A–D) ⇓ indicate that total cofilin was indeed lost from lamellipodia and cellular projections when cells were treated with the angiogenesis inhibitors. Similar results in cofilin’s subcellular localization were seen when HDMVE were treated with thrombospondin-1 (Fig. 4, E and F) ⇓ . The similar response to thrombospondin-1 in HDMVE and HUVECs indicates that the thrombospondin-1 receptor CD36, which is expressed only in HDMVE cells (54) , does not mediate this response.

Fig. 4.
Fig. 4.

Cofilin localization changes in response to fumagillin. HUVE (A–D) or HDMVE (E and F) cells were treated with 0.1 μm fumagillin (B and D) or 20 μg/ml thrombospondin-1 (F) for 24 h or left untreated (A, C, and E). Cells were fixed with formaldehyde and stained using either rabbit polyclonal anticofilin (A, B, E, and F) or rabbit polyclonal antiphospho-cofilin/ADF (C and D), followed by antirabbit Bodipy FL (Molecular Probes). Similar results were seen when cells were treated with 0.2 μm TNP-470. The results are representative of two (HDMVE) or three (HUVE) independent experiments.

Furthermore, phospho-cofilin/ADF, which was limited to the cell nucleus in untreated HUVECs, became diffusely distributed throughout the cell after fumagillin treatment (Fig. 4, C and D) ⇓ . Cofilin was localized by Western blot in both the cytosolic and nuclear fraction, and no change was detected between untreated cells and those treated with angiogenesis inhibitors (data not shown). The apparent loss of nuclear localization of the phosphorylated form seen here (Fig. 4, C and D) ⇓ may simply reflect increased phosphorylation of cytosolic cofilin. This altered regulation of subcellular localization of both the active and inactive forms of cofilin indicates actin turnover may be affected by fumagillin. Similar results were seen when HUVECs were treated with 0.2 μm TNP-470, 50 μg/ml EMAP II, or 20 μg/ml thrombospondin-1 (results not shown).

Actin Stress Fibers and Focal Adhesions Are Enhanced in Response to Angiogenesis Inhibitors.

Regulation of both cofilin and hsp27 can alter cytoskeletal dynamics. To determine the effect of angiogenesis inhibitors on the actin cytoskeleton, we treated HUVECs with fumagillin, thrombospondin-1, or EMAP II. Cells were stained with fluorescently labeled phallacidin to label F-actin. Actin stress fibers stained more densely in all of the angiogenesis inhibitor-treated cells (Fig. 5, B, D, F, and H) ⇓ . The number of cellular projections ending in lamellipodia was reduced in angiogenesis inhibitor-treated samples compared with untreated samples (Fig. 5I) ⇓ , indicating that the treated cells were in a less migratory state. Focal adhesions, the sites where stress fibers connect with the extracellular matrix, are indicative of tightly attached cells and can be visualized by staining for one of their primary components, such as the cytoplasmic focal adhesion protein, paxillin (55 , 56) . HUVECs treated with fumagillin, thrombospondin-1, or EMAP II showed many punctate paxillin foci, indicative of many focal adhesions. This was in contrast to untreated cells, whose paxillin staining at focal adhesions was sparse and faint (Fig. 5, A, C, E, G, and J) ⇓ . Similar results were seen with 0.2 μg/ml endostatin, although to a lesser degree (data not shown). These changes in stress fibers and focal adhesions were evident at 1–2 h after treatment (data not shown) and subsisted for ≤24 h (Fig. 5) ⇓ .

Fig. 5.
Fig. 5.

Actin stress fibers and focal adhesions are enhanced by angiogenesis inhibitors. HUVECs were treated with 0.1 μm fumagillin (C and D), 20 μg/ml thrombospondin-1 (E and F), 50 μg/ml EMAP II (G and H), or left untreated (A and B) for 24 h. Cells were fixed with formaldehyde and stained either for F-actin (B, D, F, and H) or paxillin (A, C, E, and G). The results are representative of at least three independent experiments. I and J, HUVE or HDMVE cells were treated as in A–H (UT, untreated; F, 0.1 μm fumagillin; TSP-1, 20 μg/ml thrombospondin-1; EMAP, 50 μg/ml EMAP II). I, actin-stained HUVE or HDMVE cells were counted, and the percentage of cells with projections ending in lamellipodia was determined for each treatment. The graph represents two independent experiments per treatment, where ≥500 cells were counted. Only cells in areas of 50–80% confluence were included in these experiments. In J, paxillin plaques seen at ×600 magnification in paxillin-stained HUVE or HDMVE cells were counted. The graph represents the number of plaques/cell in 36 cells from two independent experiments for HUVECs and 12 cells from one experiment for HDMVECs. Similar results in both actin stress fibers and focal adhesions were seen at 1–2 h after treatment (data not shown).

To determine whether the actin cytoskeletal response to angiogenesis inhibitors observed in the large vessel HUVECs is shared by CD36-expressing microvascular cells, we treated HDMVE cells with fumagillin and thrombospondin-1. As shown in Fig. 5, I ⇓ and J, the inhibitors decreased the number of lamellipodia and increased the number of focal adhesions in microvascular as well as HUVECs. These data provide evidence that alteration of the actin cytoskeleton to strengthen endothelial cell attachment to the extracellular matrix is a common mechanism for inhibition of angiogenesis by these agents.

DISCUSSION

Angiogenesis inhibitors, because of their diversity in structure and receptors used, are likely to act via distinct signaling pathways. Some of these probably control effector pathways unique to each inhibitor. However, other effector pathways are similarly modulated by many angiogenesis inhibitors. End points of these pathways would be expected to embody the most central and essential molecular targets for inhibition of angiogenesis. For this reason, we identified proteins whose abundance or isoelectric point, as seen on two-dimensional gels, was consistently changed in response to a diverse panel of potent angiogenesis inhibitors.

Although cytoskeletal organization and phosphorylation of the cytoskeletal regulatory proteins cofilin and hsp27 were coordinately altered by all of the angiogenesis inhibitors, we do not know that any of these changes directly mediate inhibition of angiogenesis by these agents. Previous studies have suggested that thrombospondin-1 and platelet endothelial cell adhesion molecule-1 coordinately regulate a series of molecular markers representative of angiogenic versus differentiated endothelial cell phenotypes (57) . On the basis of the current data, some cytoskeletal regulators exhibited disparate responses to angiogenesis inhibitors, whereas cofilin and hsp27 were consistently regulated by these inhibitors. Thus, these two proteins may be useful starting points for identifying the molecular regulatory pathways through which angiogenesis inhibitors control the angiogenic switch.

Hsp27 is phosphorylated by mitogen activated protein kinase-activated protein kinase-2 (MAPKAP-2) in response to p38 activation (58) . However, the effect of hsp27 phosphorylation on actin polymerization is controversial (Ref. 37 and references therein). Hsp27 localizes to cellular membranes under certain conditions that impact cytoskeletal dynamics (59, 60, 61) . In response to VEGF, a proangiogenic factor, actin stress fibers and focal adhesions are formed within 15 min in HUVECs, and this is accompanied by activation of p38 and phosphorylation of hsp27 under serum-free conditions (38 , 62) . In apparent contrast, we observed some of the same changes in response to angiogenesis inhibitors. Phosphorylation of hsp27 was accompanied by an increase in actin stress fibers and paxillin plaques that lasted for ≥24 h. When one considers that the actin cytoskeleton and its associated focal adhesion proteins are dynamic in nature and in constant flux when cells are moving, however, it is not surprising that similar changes are seen in these structures when cell motility is either positively or negatively impacted. Polymerization and depolymerization of actin, as well as assembly and disassembly of focal adhesions, are required for cellular motility (reviewed in Refs. 63 and 64 ). The angiogenesis inhibitors examined cause endothelial cells to adopt an adhesive state within 1–2 h after treatment (data not shown) and sustain that state for ≥24 h. Such persistent inhibition of actin dynamics may account for the inhibition of cellular motility by these agents.

Cofilin is an actin-depolymerizing protein that is inactivated in response to phosphorylation on Ser3. Active cofilin is required for cellular processes involving actin dynamics (40) . Deletion of the Drosophila ortholog of cofilin inhibits cell motility in the developing embryo (42) . Furthermore, phosphorylation of cofilin at serine 3 prevents lamellipodia extension in mammalian cells (44) . Cofilin phosphorylation and inactivation can occur through either LIM kinase or testis-specific kinase (testicular protein kinase), the latter inducing the formation of actin stress fibers (65) . The LIM kinase-dependent phosphorylation of cofilin occurs as a result of activation of the small GTPase, RhoA (66) , whose inactivation inhibits angiogenesis in vitro (67) . Phosphorylation of cofilin has been shown previously to inhibit actin dynamics, induce prominent actin stress fibers, and prevent the formation of lamellipodia in metastatic mammary adenocarcinoma cells (44 , 66) . In both large and small vessel endothelium, all of the angiogenesis inhibitors that we tested coordinately regulated cofilin phosphorylation and increased both the density of actin stress fibers and number of focal adhesion plaques as measured by paxillin staining. These data suggest that cofilin and the actin cytoskeleton are principal targets for inhibition of angiogenesis.

Some effects of individual angiogenesis inhibitors on the endothelial cytoskeleton have been reported previously. In murine brain endothelial cells, endostatin causes a change in cell shape characterized by a flattening of the cell body and decrease in cell projections (68) . In contrast, both thrombospondin-1 and endostatin cause actin stress fibers to become sparse and focal adhesion plaques less numerous (56 , 69 , 70) . The apparent discrepancy with our present data may arise from differences in the cell culture conditions before and during treatment. In the previous work, endothelial cells were grown to 80% confluence with limiting serum, possibly causing them to adopt a quiescent phenotype. Under such conditions, cells are in a state of tight adhesion. The addition of endostatin or thrombospondin-1 to the cells caused them to revert to a deadhesive state (56 , 69, 70, 71) , characterized by a lower density of actin stress fibers and fewer focal adhesions.

These contradictory effects of thrombospondin-1 on endothelial cell adhesion may also be explained by the presence of several thrombospondin-1 receptors on these cells. Thrombospondin-1 inhibits endothelial cell chemotaxis through its receptors CD36 (72) and heparan sulfate proteoglycan (73) but stimulates chemotaxis of endothelial cells through α6β1 integrin (74) . Integrins promote endothelial cell adhesion to thrombospondin-1 (74 , 75) , but calreticulin mediates a deadhesive signal from the same protein (76) . Differential expression or activation of these receptors may, therefore, account for the different cytoskeletal responses reported here and elsewhere. Here, we show that cofilin phosphorylation and actin cytoskeleton organization are induced by thrombospondin-1 in both HDMVEC (CD36+) and HUVEC (CD36−). Thus, CD36 is not required for this response. The roles of other thrombospondin-1 receptors in regulation of these responses remain to be defined.

The initial condition of the cells used in this study more closely resembles that of angiogenic endothelium, because serum growth factors were included, and the cells were actively proliferating and competent to migrate. After treatment with angiogenesis inhibitors, the cells developed a dense network of actin stress fibers terminating in many focal adhesions, characteristic of quiescent cells that cannot migrate. The effect of the angiogenesis inhibitors on the cytoskeleton persisted for ≥24 h. This sustained increase in abundance of stress fibers and focal adhesions conveys a state of a rigidity that is characteristic of nonmotile cells. This is further supported by the observed decrease in lamellipodia after treatment with angiogenesis inhibitors.

Inhibiting actin dynamics is a plausible target for angiogenesis inhibitors. Controlled cytoskeletal remodeling is essential for cell motility and division, both processes that are required for angiogenesis. Inhibition of capillary endothelial cell spreading or actin dynamics prevents passage through G1 phase, synthesis of cyclin D1, degradation of p27, and phosphorylation of the retinoblastoma gene product (77) . The fact that the cytoskeletal modulators cofilin and hsp27 are both modified in response to all of the angiogenesis inhibitors tested here indicates that the function of these proteins is of general importance for angiogenesis. Treatment of cells in culture with antiangiogenic compounds may constitute a type of cellular “stress.” However, others have shown that both hyperthermia and osmotic shock cause decreases in both actin stress fiber and focal adhesion density (78 , 79) , indicating that the response seen in this study is not a simple reaction of cells to stress. Future studies might address further the cellular specificity of these inhibitors’ effects on cytoskeletal architecture. It is plausible that the endothelial cytoskeleton is modified in response to distinct signaling pathways and that these cells may be uniquely affected by a particular inhibitor of actin dynamics. The common morphology seen in angiogenesis inhibitor-treated endothelial cells may represent convergence of the signaling pathways modulated by the inhibitors used in this study. Identification of the mechanisms by which angiogenesis inhibitors regulate this end point will increase our understanding of the angiogenic phenotype. The possibility of specifically targeting endothelial cells for the inhibition of migration and/or proliferation through actin dynamics would be an interesting avenue to explore for in vivo therapies.

In a broader context, these data show that a proteomic approach can be used to identify central molecular targets for a biological modifier. Angiogenesis is fundamental to tumor progression, but the inhibitors available at this time have mixed effects on cells in culture and have been limited in their success in cancer patients (6) . For this reason, we must identify specific receptors and downstream targets that mediate their inhibition of tumor growth. In this study, we used a proteomics strategy to identify molecules and examine effects shared by distinct angiogenesis inhibitors. We are currently extending this work to explore the functions of more protein targets differentially expressed or modified in endothelial cells in response to these inhibitors.

Acknowledgments

We thank J. R. Bamburg for the generous gift of antiphospho-cofilin/ADF polyclonal antibody, EntreMed (Rockville, MD) for providing endostatin, and C. Haggerty and K. Gardner for valuable advice regarding subcellular protein extraction.

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 a National Cancer Institute Intramural Research Award (S. M. K.) and by an award from the National Cancer Institute Breast Cancer Faculty (S. E. I.).

  • ↵2 To whom requests for reprints should be addressed, at Laboratory of Pathology, National Cancer Institute, NIH, Bethesda, MD 20892. Phone: (301) 496-6264; Fax: (301) 402-0043; E-mail: droberts{at}helix.nih.gov

  • ↵3 The abbreviations used are: EMAP, endothelial monocyte activating polypeptide; VEGF, vascular endothelial growth factor; hsp, heat shock protein; HUVEC, human umbilical vein endothelial cell; BCA, bicinchoninic acid; HRP, horseradish peroxidase; DPBS, Dulbecco’s phosphate buffered saline.

  • ↵4 S. E. Ivie and D. D. Roberts, unpublished data.

  • Received December 4, 2002.
  • Revision received July 15, 2003.
  • Accepted July 22, 2003.

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Cancer Research: 63 (19)
October 2003
Volume 63, Issue 19

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Angiogenesis Inhibitors Target the Endothelial Cell Cytoskeleton through Altered Regulation of Heat Shock Protein 27 and Cofilin
Susan M. Keezer, Susan E. Ivie, Henry C. Krutzsch, Anita Tandle, Steven K. Libutti and David D. Roberts
Cancer Res October 1 2003 (63) (19) 6405-6412;
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