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(The FASEB Journal. 2000;14:2589-2600.)
© 2000 FASEB

Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5

JING-CHUAN ZHANG*, KEVIN CLAFFEY{ddagger}, RAMASAMY SAKTHIVEL*,{dagger}, ZBIGNIEV DARZYNKIEWICZ§, DAVID ELLIOT SHAW, JUAN LEAL{dagger}{dagger}, YI-CHUN WANG{dagger}{dagger}, FENG-MIN LU{ddagger}{ddagger} and KEITH R. MCCRAE*,{dagger}1

* Hematology-Oncology Division and
{dagger} Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA;
{ddagger} Department of Physiology, Center for Vascular Biology, University of Connecticut School of Medicine, Farmington, Connecticut, USA;
§ New York Medical College, Valhalla, New York, USA;
D.E. Shaw & Co., Inc., New York, New York and Attenuon L.L.C, San Diego, California, USA;
{dagger}{dagger} Department of Chemotherapeutics, Pharmaceutical Product Division, Abbott Laboratories, Abbott Park, Illinois, USA; and
{ddagger}{ddagger} Center for Neurovirology and Neurooncology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania, USA

1Correspondence: Hematology-Oncology Division, BRB 3, Case Western Reserve University, School of Medicine, 10900 Euclid Ave., Cleveland, OH 44106-4937, USA. E-mail: kxm71{at}po.cwru.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We previously reported that the binding of two-chain high molecular weight kininogen (HKa) to endothelial cells may occur through interactions with endothelial urokinase receptors. Since the binding of urokinase to urokinase receptors activates signaling responses and may stimulate mitogenesis, we assessed the effect of HKa binding on endothelial cell proliferation. Unexpectedly, HKa inhibited proliferation in response to several growth factors, with 50% inhibition caused by ~10 nM HKa. This activity was Zn2+ dependent and not shared by either single-chain high molecular weight kininogen (HK) or low molecular weight kininogen. HKa selectively inhibited the proliferation of human umbilical vein and dermal microvascular endothelial cells, but did not affect that of umbilical vein or human aortic smooth muscle cells, trophoblasts, fibroblasts, or carcinoma cells. Inhibition of endothelial proliferation by HKa was associated with endothelial cell apoptosis and unaffected by antibodies that block the binding of HK or HKa to any of their known endothelial receptors. Recombinant HK domain 5 displayed activity similar to that of HKa. In vivo, HKa inhibited neovascularization of subcutaneously implanted Matrigel plugs, as well as rat corneal angiogenesis. These results demonstrate that HKa is a novel inhibitor of angiogenesis, whose activity is dependent on the unique conformation of the two-chain molecule.—Zhang, J.-C., Claffey, K., Sakthivel, R., Darzynkiewicz, Z., Shaw, D. E., Leal, J., Wang, Y.-C., Lu, F. M., McCrae, K. R. Two-chain high molecular weight kininogen induces endothelial cell apoptosis and inhibits angiogenesis: partial activity within domain 5.


Key Words: angiogenesis • coagulation • neovascularization • tumor • endothelium


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
RECENT STUDIES HAVE demonstrated the importance of angiogenesis in several biological processes (1) . Of particular interest is the observation that the growth of tumors is angiogenesis dependent (1 , 2) . Primary or metastatic tumor foci are unable to achieve a size of more than several millimeters in the absence of neovascularization (1 , 3) . Serial evaluation of transgenic mice predisposed to develop neoplasms has demonstrated that neovascularization of premalignant lesions precedes their evolution into tumors (2) and that inhibition of angiogenesis delays the growth of such lesions (4) . In humans, increased microvessel density is associated with a poor prognosis in a number of tumor types (5 6 7) .

An emerging paradigm is that proteolytic fragments or conformationally altered forms of specific plasma or extracellular matrix proteins inhibit angiogenesis (2 , 8) . Several of these are members of the coagulation and/or fibrinolytic systems. Examples include angiostatin, which contains kringles 1–4 of plasminogen (9) , prothrombin kringle 2 (10) , plasminogen activator inhibitor type 1 (PAI-1) (11) , and antithrombin (12) . Thrombospondin, an abundant component of platelet alpha granules, as well as peptides derived from its procollagen domain and properdin-like type 1 repeats, also inhibit angiogenesis (13) . A partial list of other antiangiogenic polypeptides includes endostatin, a 20 kDa carboxyl-terminal fragment of collagen XVIII (14) , the related collagen XV fragment, restin (15) , the hemopexin domain of matrix metalloprotease 2 (16) , an amino-terminal fragment of prolactin (17) , a 29 kDa fibronectin fragment (18) , a 24 kDa fragment of the {alpha}2 chain of type IV collagen (19) , and vasostatin, an amino-terminal fragment of calreticulin (20) .

High molecular weight kininogen (HK) is a ~120 kDa single-chain glycoprotein that plays a central role in contact activation (21) . HK is comprised of heavy and light chains that contain domains 1 through 3, and 5 and 6, respectively, and are linked by domain 4, which contains the vasoactive nonapeptide bradykinin (22) (Fig. 1 ). Cleavage of HK between Lys362-Arg363 and Arg371-Ser372 by kallikrein (23) , an event that may occur on the endothelial surface (24) , results in the release of bradykinin and generation of two-chain high molecular weight kininogen (HKa). HKa contains a 62 kDa heavy chain and 56 kDa light chain linked by a single disulfide bond (25) . Kallikrein may further cleave HKa between Arg419-Lys420, reducing the size of the light chain to ~46 kDa (Fig. 1) (25) . Conversion of HK to HKa is accompanied by a dramatic conformational rearrangement characterized by reorientation of HKa domains and increased exposure of domain 5 (21) . Another member of the kininogen family, low molecular weight kininogen (LK), is comprised of a heavy chain identical to that of HKa, bradykinin, and a unique 4 kDa light chain (D5L) (Fig. 1) (26) . In this report we demonstrate that HKa, but not HK or LK, induces endothelial apoptosis and inhibits angiogenesis in vivo.



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  		Figure 1. Domain structure of high and low molecular weight kininogens. Single-chain high molecular weight kininogen (HK) consists of 5 domains, with the light chain comprised of domains 1–3, and the heavy chain of domains 5 and 6. The heavy and light chains are linked by domain 4, which contains the nonapeptide bradykinin (RPPGFSPFR). Bradykinin is released after cleavage of HK by kallikrein (or other enzymes, see Discussion), with the resultant generation of two-chain high molecular weight kininogen (HKa) in which the heavy and light chains are linked by a single disulfide bond between Cys10 of domain 1 and Cys596 of domain 6. The NH2 terminus of the light chain may begin with either Ser372 or, if further processed, Lys420. Low molecular weight kininogen (LK) is comprised of a heavy chain identical to that of HK and HKa, but a unique 4 kDa light chain (D5L) derived by alternative splicing.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tissue culture medium was from Mediatech (Herndon, Va.) and fetal bovine serum from Hyclone (Logan, Utah). Recombinant human basic fibroblast growth factor (bFGF), vascular endothelial cell growth factor (VEGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) were from Collaborative Biomedical Products (Bedford, Mass.). NHS-LC biotin and Super Signal chemiluminescence reagent were from Pierce (Rockford, Ill.). Gelatin, hydron, streptavidin-peroxidase and rabbit anti-bradykinin antiserum was from Sigma (St. Louis, Mo.). Single and two-chain high molecular weight kininogen were from Enzyme Research Labs (South Bend, Ind.). HK was >99% single chain, as determined by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after reduction. The NH2 termini of the HKa heavy and light chains were determined by amino acid sequencing on an Applied Biosystems Procise sequencer. These studies did not reveal a heavy chain sequence, demonstrating that the NH2 terminus of the HKa heavy chain was blocked, and the remainder of the heavy chain intact. Major and minor light chain sequences were obtained beginning, respectively, with Lys420 and Ser372. These NH2 termini are consistent with the known kallikrein cleavage sites within HK (21 , 25) , as well as the presence of predominant ~62 kDa and ~46 kDa bands upon analysis of HKa by 10% SDS-PAGE under reducing conditions. LK was from American Research Products (Belmont, Mass.). Bradykinin was from Peninsula (Belmont, Calif.). All HKa preparations contained <0.05 ng/ml of endotoxin, determined using the E-Toxate (Limulus amoebocyte lysate) assay (Sigma).

Cell culture and endothelial cell proliferation assays
Human umbilical vein endothelial cells (HUVEC), smooth muscle cells, and fibroblasts, as well as human aortic smooth muscle cells were cultured and characterized as described previously (27 , 28) . Human dermal microvascular endothelial cells (HDMVEC) were obtained from Technoclone (Vienna, Austria). Human trophoblast cells (ED 27) (29) were provided by Dr. Douglas Kniss, Department of Obstetrics and Gynecology, Ohio State University School of Medicine. HEK 293 cells, MDA MB-231 breast carcinoma cells and U937 cells were obtained from the ATCC (Rockville, Md.).

To assess the effect of HKa on endothelial cell proliferation, cells were suspended at a concentration of 3 x 104 cells/ml in medium 199 (M199) containing 2% fetal calf serum (FCS). One hundred microliters of this suspension was plated in individual wells of a 96-well microplate precoated with 1% gelatin (or, in selected experiments, other extracellular matrix proteins). After incubation for 4 h to allow cells to adhere and spread, medium was replaced with fresh M199 containing 2% FCS (or, in selected experiments, 10% human AB serum), 10 ng/ml bFGF (or another growth factor, as specified), 10 µM ZnCl2, and, in experimental wells, HKa. Cells were then incubated for 48 h at 37°C, at which time the relative numbers of cells in each well were determined using the Cell Titer AQueous cell proliferation assay (Promega, Madison, Wis.). The percent inhibition of proliferation by HKa was determined using the formula:

where (+GF) and (-GF) represent proliferation in the presence or absence, respectively, of growth factor, and (+GF, HKa) represents proliferation in the presence of both growth factor and HKa.

To determine whether HK was converted to HKa during the course of endothelial cell proliferation assays, HK was labeled with NHS-LC biotin, as described (30) . Labeled HK was subjected to 10% SDS-PAGE under reducing conditions and transferred to a polyvinylidene fluoride membrane. The immobilized protein was detected by incubation of the membrane with a 1:500 dilution of streptavidin-peroxidase, followed by exposure to Super Signal chemiluminescence reagent. HK remained > 99% single chain after labeling. The ability of biotinylated HK to inhibit endothelial cell proliferation was then assessed, with the extent of conversion of the biotin-HK to biotin-HKa during the assay determined simultaneously by analysis of conditioned medium and cell extracts obtained 24 and 48 h after addition of biotin-HK to cells. Parallel studies in which biotin-HK was incubated in fresh, cell-free medium were also performed. Bands observed on the autoradiograms were quantitated using NIH Image v 1.59. In complementary studies, we determined that both the heavy and light chains of HKa were labeled with NHS-LC biotin, that the antiproliferative activity of HKa was not affected by biotinylation, and that HKa was not further degraded during the course of proliferation assays.

In selected experiments, we assessed the ability of antibodies that block the binding of HK or HKa to specific endothelial cell receptors to directly inhibit endothelial cell proliferation or to inhibit the antiproliferative activity of HKa. Three antibodies were used. An affinity-purified rabbit polyclonal antibody that inhibits the binding of HKa to a site within domains 2 + 3 of the urokinase receptor has been described (28) . A monoclonal antibody that blocks the binding of HK to the receptor for the globular heads of C1q (gC1qR) was a kind gift from Dr. Berhane Ghebrehewit, SUNY, Stony Brook (31) . A rabbit antiserum that blocks the binding of HK to an additional endothelial cell HK receptor, cytokeratin 1, as well as a 15 amino acid peptide that corresponds to the HK binding region within cytokeratin 1 and inhibits HK binding to this receptor (IC50 ~10 µM), were kindly provided by Dr. Alvin Schmaier, University of Michigan (32 , 33) .

Detection of endothelial cell apoptosis
Endothelial cell apoptosis after exposure of cells to HKa was documented using several assays. First, endothelial cells plated on gelatin-coated glass coverslips were cultured in medium 199 containing 2% FCS in the absence or presence of 30 nM HKa for 6–24 h. Cells were then fixed in phosphate-buffered saline (PBS) containing 1% formaldehyde and stained with a solution containing 1 µg/ml of 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI) and 10 µg/ml of sulforhodamine 101 (Molecular Probes, Eugene, Oreg.). Stained cells were visualized by UV illumination using a Nikon Microphot FXA microscope (objective 40x, Neofluor). To assess DNA fragmentation after exposure of cells to HKa, cytoplasmic DNA was isolated from control and HKa-exposed cells using a buffer containing 0.4% Triton X-100, and analyzed by 0.8% agarose gel electrophoresis and ethidium bromide staining. Apoptosis was also assessed by flow cytometric analysis of control and HKa-exposed cells labeled with fluorescein-dUTP using the TUNEL reaction (In Situ Cell Death Detection Kit, Boehringer Mannheim, Indianapolis, Ind.).

Inhibition of angiogenesis by HKa
The effect of HKa on angiogenesis in vivo was determined using the Matrigel plug and corneal micropocket angiogenesis assays. In the former, athymic Ncr nude mice (7–8 wk, female) were injected subcutaneously (s.c.) with 0.25 ml of chilled Matrigel containing 400 ng of bFGF and 25 µg heparin, to which either 25 µl of PBS (left flank injection) or an equal volume of PBS containing 10 µg HKa (right flank injection) had been added. The Matrigel solidified after injection and remained as an intact, s.c. plug for the duration of the experiment. Mice were killed after 4 days, and digital images of intact control and experimental plugs were obtained prior to en bloc excision of the plugs and overlying skin for histological analysis (21) .

The corneal micropocket angiogenesis assay was performed as described previously (34 , 35) . Pellets were prepared using 12% hydron. Control pellets containing 50 ng of bFGF were implanted in the left eyes of 10 Sprague-Dawley rats. Test pellets containing 50 ng of bFGF and 12 ng of HKa were implanted in the right eyes of the same animals. Each pellet was implanted in a 2 mm pocket prepared in the cornea, 1 mm from the limbus. Corneal neovascularization was assessed 7 days after implantation of the pellets, at which time digital images of each eye were obtained using a Nikon NS-1 slit lamp. The total area of neovessels in each digital image was then determined using a Leica-Qwin (Northvale, N.J.) image analysis system (36) .

Preparation of recombinant HK domain 5 (HK-D5)
The exposure of HKa domain 5, which contains high-affinity endothelial cell binding sites (37) , is enhanced after cleavage of HK to HKa (38) . To determine whether domain 5 might mediate the effects of HKa on endothelial cells, a 301 nucleotide cDNA encompassing nucleotides 173–472 of kininogen exon 10 and encoding amino acids 412–503 of kininogen was generated by reverse transcription-polymerase chain reaction (RT-PCR) of human liver RNA. Amplification was performed in a 100 µl reaction mixture containing 2.5 U Pfu DNA polymerase (Stratagene, La Jolla, Calif.), 10 µl 10x Pfu buffer, 0.8 µl of 100 mM dNTPs, 5 µl first strand reaction mix, and 100 ng each of sense and antisense primers. The sense primer was 5'-CAGGGATCCAAAATGGACTGGGGCCATGAAAAA-3', and the antisense primer was 5'-GGCGAATTCAGAAGAGCTTGCCAAATG-3'. The PCR product was cloned into pGEX-6p-1 (Pharmacia Biotech, Piscataway, N.J.). This polypeptide encompassed a previously defined high-affinity endothelial cell binding region (amino acids 479–498) within HK domain 5 (30) . The glutathione-S-transferase (GST) domain 5 fusion protein was isolated using glutathione-agarose, and GST-free domain 5 was purified by a second passage of the PreScission protease (Pharmacia-Biotech) -cleaved fusion protein over the same column. Purified domain 5 migrated with the expected Mr of ~11.1 when analyzed by 16% tricine SDS-PAGE and yielded a single peak when analyzed by reverse-phase high performance liquid chromatography (HPLC). Amino-terminal amino acid sequencing of recombinant domain 5 was ambiguous beyond 9 amino acids. However, the first 6 amino acids corresponded to vector-encoded sequence carboxyl-terminal to the PreScission protease cleavage site, whereas the next three amino acids were identical to the kininogen sequence beginning at Asp412. Recombinant domain 5 preparations were routinely passed through a Detoxigel column (Pierce) before study, after which they contained < 0.05 ng/ml of endotoxin as determined using the E-Toxate assay (Sigma).

Free GST was prepared from a control pGEX-6p-1 vector and used as a negative control in studies where the effect of free domain 5 on endothelial cell proliferation was assessed. The 39 kDa low density lipoprotein receptor-related protein/{alpha}2-macroglobulin receptor receptor-associated protein (RAP) was also prepared as a GST fusion protein. GST-free RAP was isolated as described (39) and used as an additional control for studies utilizing HKa domain 5.

Synthetic peptides
Synthetic 16 amino acid peptides, each overlapping by 8 amino acids, were prepared based on the amino acid sequence of HK domain 5 (amino acid residues 384–503 of HK). Peptides were synthesized on a Rainin Symphony peptide synthesizer, using standard solid-phase methods (40) . FMOC amino acids were purchased from Perseptive Biosystems (Foster City, Calif.). All peptides were purified using reverse-phase HPLC and purity was analyzed by MALDI-TOF mass spectrometry.

Statistics
Error bars in all figures represent the standard deviation of quadruplicate points. Each experiment was performed a minimum of three times. The significance of differences in the numbers of endothelial cells remaining in each well at the end of proliferation assays, as well as that between the total vessel area in rat corneas that received hydron pellets containing bFGF or bFGF and HKa, was determined using the Student’s two-tailed t test for paired samples.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HKa inhibits endothelial cell proliferation in a zinc-dependent manner
Previous studies from our laboratory demonstrated that HKa may bind to endothelial cells through interactions with the urokinase receptor (uPAR) (28) . Since ligand binding to uPAR has been associated with activation of signaling events (41 42 43) and stimulation of cell proliferation (44) , we wished to determine the effect of HKa binding on the proliferation of endothelial cells. Contrary to expectations, HKa potently inhibited the proliferation of HUVEC and HDMVEC. This activity was dependent on supplementation of the medium with ZnCl2 (Fig. 2 ), perhaps reflecting the requirement for Zn2+ for specific binding of HKa to endothelial cells (45) . Maximal effects occurred at a Zn2+ concentration of 10 µM, demonstrating that HKa inhibits endothelial proliferation at physiological (<15 µM) Zn2+ concentrations. In the absence of HKa, Zn2+ did not affect endothelial cell proliferation.



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  		Figure 2. Effect of Zn2+ on inhibition of endothelial cell proliferation by HKa. HUVEC (3x104 cells/ml) were plated in gelatin-coated 96-well microplates and cultured for 4 h at 37°C. Medium was then replaced with fresh medium 199 containing 2% fetal bovine serum, 10 ng/ml bFGF, 30 nM HKa, and either no or increasing concentrations of ZnCl2. After 48 h, the relative numbers of cells in each well and the percent inhibition of proliferation caused by HKa were determined.

Inhibition of endothelial cell proliferation is mediated selectively by HKa
HKa inhibited endothelial cell proliferation by 50% at a concentration of ~10 nM (Fig. 3 ). In contrast, LK, bradykinin, or anti-bradykinin antibodies did not affect endothelial cell proliferation. HK (single chain) inhibited proliferation only modestly at concentrations approaching 100 nM (Fig. 3) , with 50% inhibition evident at a concentration of ~320 nM. However, studies using biotinylated HK revealed that ~2% of the added HK was converted to HKa during the course of the proliferation assays (not shown). Since biotinylation did not affect the antiproliferative activity of HKa, it is likely that the newly generated HKa contributed to the observed activity of HK. Hence, these studies suggest a critical role for the unique conformation of the two-chain HKa molecule in mediating the antiproliferative activity of high molecular weight kininogen.



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  		Figure 3. Inhibition of endothelial cell proliferation by HKa, HK, and low molecular weight kininogen (LK). HUVEC (3x104 cells/ml) were plated in gelatin-coated 96-well microplates and cultured for 4 h at 37°C. Medium was then replaced with fresh medium 199 containing 2% fetal bovine serum, 10 ng/ml bFGF, 10 µM ZnCl2, and either no kininogen or increasing concentrations of HKa (black bars), HK (white bars), or LK (striped bars). After 48 h, the relative numbers of cells in each well and the percent inhibition of proliferation caused by each of the kininogen species were determined.

Characterization of the antiproliferative activity of HKa
The antiproliferative activity of HKa was apparent within 6 h of its addition to cells and was maintained in the presence of 10% human serum. Extensive dialysis (25,000 Mr cutoff) did not affect the activity of HKa. HKa completely inhibited endothelial proliferation in response to several growth factors, including bFGF, VEGF, HGF, and PDGF (not shown). Since the mitogenic activity of each of these is mediated through interactions with distinct receptors, we hypothesize that the mechanism(s) by which HKa inhibits endothelial proliferation is unlikely to depend entirely on inhibition of growth factor binding.

HKa inhibited the proliferation of human umbilical vein and microvascular endothelial cells with similar potency, though it did not affect the proliferation of several other cell types, including primary cultures of human aortic smooth muscle and trophoblast cells (Table 1 ). Moreover, in studies in which endothelial cells, smooth muscle cells and fibroblasts were isolated from the same umbilical cord, we confirmed that the effects of HKa were endothelial cell specific (Table 1) . The ability of HKa to inhibit endothelial cell proliferation was inversely proportional to cell density, with no effect on confluent or near-confluent endothelial cells.


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  		Table 1. Inhibition of endothelial cell proliferation by HKa (30 nM)a

In selected experiments, we assessed the ability of HKa to inhibit the proliferation of endothelial cells on different extracellular matrix (ECM) proteins. HKa (10 nM) potently inhibited the proliferation of HUVEC cultured on gelatin, laminin, and Matrigel though a slightly less potent inhibition, overcome by a higher concentration of HKa (50 nM), occurred when cells were cultured on fibronectin or vitronectin. Intermediate effects were observed when cells were cultured on fibrinogen, and cells cultured on collagen types I or IV were relatively resistant to the antiproliferative activity of HKa.

To define the endothelial cell receptors that mediate the effects of HKa, we assessed whether its antiproliferative activity was inhibited by antibodies that inhibit the binding of HK or HKa to specific endothelial cell receptors (Table 2 ). Neither an affinity-purified polyclonal antibody that blocks the binding of HKa to endothelial cell urokinase receptors (28) , a monoclonal antibody that blocks the binding of HK to endothelial cell gC1q receptors (31 , 46) , nor a polyclonal antibody that blocks HK binding to endothelial cell CK-1 (32 , 33) affected the antiproliferative activity of HKa. Moreover, a 15 amino acid peptide (PGG-15) from within the HK binding region of cytokeratin 1, which inhibits the binding of HK to cytokeratin-1 by 50% at a concentration of 9–10 µM (33) , did not affect the antiproliferative activity of HKa even when used at concentrations exceeding 250 µM (not shown).


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  		Table 2. Inhibition of endothelial cell proliferation by HKa and HK/HKa receptor antibodies

The antiproliferative activity of HKa is associated with endothelial cell apoptosis
To determine whether inhibition of endothelial cell proliferation by HKa was associated with endothelial cell apoptosis, we stained control and HKa-exposed cells with DAPI, observing morphological changes characteristic of apoptosis (nuclear condensation, hyperchromaticity, and fragmentation) in 30–50% of HKa-exposed cells within 6 h of HKa addition (Fig. 4 ). These changes were apparent in all of the cells at later time points. Consistent with these results, a specific ‘laddering’ pattern of DNA fragmentation, characteristic of apoptosis, was apparent on electrophoretic analysis of DNA from cells exposed to HKa (47) (Fig. 5 ); flow cytometric analysis of control and HKa-exposed cells labeled with fluorescein-dUTP using the TUNEL reaction revealed increased labeling of HKa-exposed cells. HKa-exposed cells did not stain with trypan blue, demonstrating that HKa did not cause cell lysis or cytotoxicity.



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  		Figure 4. Induction of endothelial cell apoptosis by HKa: DAPI staining. HUVEC were plated on gelatin-coated glass coverslips at a density of 3 x 104 cells/ml. Medium was replaced with fresh medium 199 containing 2% fetal bovine serum, 10 ng/ml bFGF, 10 µM ZnCl2, and either 30 nM HKa or no HKa. After culture for increasing intervals, cells were stained with DAPI and sulforhodamine. A) Cells cultured for 16 h in the absence of HKa. B) Cells cultured in the presence of HKa for 4 h. The four cells on the right reveal loss of the normally smooth nuclear contour, nuclear hyperchromaticity, and condensation. C) Cells cultured for 16 h in the presence of HKa.



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  		Figure 5. DNA fragmentation in endothelial cells exposed to HKa. A) HUVEC (3x104 cells/ml) were plated in gelatin-coated 12-well tissue culture plates and cultured for 4 h at 37°C. Medium was then replaced with fresh medium 199 containing 2% fetal bovine serum, 10 ng/ml bFGF, 10 µM ZnCl2, and either no kininogen or 30 nM HKa. At various intervals, cytoplasmic DNA was isolated and analyzed using 0.8% agarose gel electrophoresis. B) Cells were plated as in panel A, then incubated with either no or increasing concentrations of HKa for 6 h. DNA was then analyzed as in panel A.

HKa inhibits angiogenesis
The observations that HKa specifically induced apoptosis of subconfluent, proliferative endothelial cells suggested that it might inhibit angiogenesis. Therefore, we assessed the ability of HKa to inhibit the neovascularization of s.c.-implanted Matrigel plugs containing bFGF. As depicted in Fig. 6A (left panels), Matrigel plugs that contained bFGF induced exuberant vessel ingrowth within 4 days after implantation. In contrast, little neovascularization occurred in plugs that contained bFGF and HKa (Fig. 6A , right panels). Histological analysis of control and HKa-containing plugs confirmed that the vessel density within the latter was markedly reduced (Fig. 6B ).



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  		Figure 6. HKa inhibits the neovascularization of Matrigel plugs Matrigel ‘plugs’ were implanted s.c. Control plugs contained 400 ng/ml bFGF, 25 µg heparin, and 25 µl of PBS. Experimental plugs were identical, but also contained 10 µg of HKa. Mice were killed 4 days after implantation of the plugs. A) Left panels (12. 5x and 32x): gross appearance of the control plug (12. 5x and 32. 5x). Several neovessels (arrows) are apparent that are distinguishable from underlying, preexisting vessels in the dermis. The asterisk denotes a tortuous neovessel arising from a larger, preexisting arteriole (shown at higher resolution in the lower left panel). Right panels (12. 5x and 32x): gross appearance of the HKa-containing plug. Though underlying vessels in the dermis may be visualized through the transparent Matrigel plug, there is no neovascularization of the implant. B) Histology of the control (left panel) and HKa-containing (right panel) plugs. Numerous blood-containing vessels (arrowheads) are present in the control plug, whereas the vessel density in the HKa-containing plug is markedly reduced and blood flow has not been established.

The antiangiogenic activity of HKa was also assessed by determining its effect on rat corneal angiogenesis. In 10 control corneas, bFGF-containing hydron pellets induced a robust angiogenic response (Fig. 7A , left panel). In comparison, the length and density of neovessels were significantly reduced in 10 corneas in which the implanted pellets contained bFGF and HKa (Fig. 7B , right panel). Computer analysis of digital images revealed that the mean vessel area within corneas that received HKa-containing pellets (53,931 µm2) was reduced by 82% compared to those in which pellets contained bFGF only (293,807 µm2) (P<0.000005).



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  		Figure 7. HKa inhibits rat corneal neovascularization. Hydron pellets were implanted in rat corneas as described in Material and Methods. The control pellet (left panel) contained 50 ng bFGF, whereas the test pellet (right panel) contained 50 ng bFGF and 12 ng HKa. Corneal neovascularization was analyzed after 7 days.

HKa domain 5 inhibits endothelial cell proliferation and induces endothelial apoptosis
Previous studies have demonstrated the presence of endothelial cell binding regions within domain 5 of kininogen (30 , 37) . Since the exposure of domain 5 is enhanced on cleavage of HK (38) , we hypothesized that domain 5 might mediate the antiangiogenic activity of HKa. To address this hypothesis, we determined the effect of a recombinant domain 5 polypeptide encompassing amino acids 412–503 of kininogen on endothelial proliferation. Recombinant domain 5 potently inhibited proliferation (IC50 ~40 nM) in a Zn2+ and concentration-dependent manner (Fig. 8A , B , respectively) and, like HKa, induced endothelial cell apoptosis (not shown). Neither the isolated GST fusion partner from the GST domain 5 polypeptide nor recombinant ~39 kDa RAP affected endothelial cell proliferation (Fig. 8A ). HKa domain 5 also inhibited bFGF and VEGF-induced angiogenesis in the chick chorioallantoic membrane (not shown).



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  		Figure 8. Inhibition of endothelial cell proliferation by HK domain 5. A) HUVEC (3x104 cells/ml) were plated in gelatin-coated 96-well microplates and cultured for 4 h at 37°C. Medium was then replaced with fresh medium 199 containing 2% fetal bovine serum, 10 ng/ml bFGF, and either HKa, purified HK domain 5, purified GST, or purified 39 kDa receptor-associated protein (RAP) derived from a GST-RAP fusion protein and used as an additional control. HKa was used at a concentration of 20 nM, whereas all other proteins were used at 80 nM. The activity of each protein was assessed in the absence (white bars) or presence (black bars) of 10 µM Zn2+. B) The effect of increasing concentrations of recombinant HK domain 5 (10–640 nM) on endothelial cell proliferation in the presence of 10 µM Zn2+. HKa (20 nM) was used as a positive control.

To further define the active regions within HK domain 5, synthetic 16 amino acid peptides encompassing domain 5 (amino acids 384–503), each overlapping by 8 amino acids, were prepared and tested for their ability to inhibit endothelial cell proliferation (Table 3 ). These studies revealed activity within peptides from the carboxyl-terminal region of domain 5, with two peptides (H5–13 and H5–14) that encompass the high-affinity endothelial cell binding region within domain 5 (amino acids 479–498 of kininogen) (30) causing near-complete inhibition of proliferation when used at a concentration of 50 µM. In subsequent studies, we determined that these peptides inhibited endothelial cell proliferation by 50% at concentrations of ~8 µM and 14 µM, respectively.


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  		Table 3. Inhibition of endothelial cell proliferation by HKa domain 5 peptidesa


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
These studies demonstrate that HKa is a potent inhibitor of angiogenesis that selectively induces apoptosis of proliferating endothelial cells. The in vitro potency of HKa is comparable to that of other antiangiogenic polypeptides such as angiostatin (48 49 50) , endostatin (14) , and thrombospondin 1 (13) . Hence, when considering the abundance of HK in plasma (~670 nM), it is likely that HKa may be a physiologically important regulator of angiogenesis.

The mechanisms by which HKa causes endothelial cell apoptosis and inhibits angiogenesis have not been defined. Previous reports have demonstrated that HKa inhibits spreading of several cell types on vitronectin (51) and fibrinogen (52) . It has been proposed that these effects may result from the binding of HKa to vitronectin, thereby inhibiting the interaction between the RGD site within vitronectin and integrin {alpha}vß3 (51) . Based on this hypothesis, it is possible that HKa might induce endothelial cell apoptosis by causing disadhesion of cells from the extracellular matrix and disrupting integrin-mediated signaling (anoikis) (53 , 54) . For several reasons, however, we do not believe that this mechanism is likely to be solely responsible for the results obtained in this study. First, HKa concentrations ~10-fold higher than those that induce apoptosis (10 nM) are required to inhibit endothelial cell adhesion and spreading (51 ; J.-C. Zhang, and K. R. McCrae, unpublished results). Second, in addition to vitronectin, HKa causes apoptosis of endothelial cells cultured on several ECM substrates, including laminin, Matrigel, fibronectin, and fibrinogen. Though the ability of {alpha}vß3 to bind several ECM ligands (55 56 57) makes it difficult to exclude the possibility that the effects of HKa reflect disruption of {alpha}vß3-substratum interactions, adhesion to some of the purified ECM components used in these studies (fibrinogen, laminin) is mediated primarily by integrins other than {alpha}vß3. Furthermore, neither fibrinogen (an {alpha}vß3 ligand) nor an anti-ß3 integrin monoclonal antibody, 7E3, inhibit the binding of HKa to endothelial cells (28) . Third, the ability of HKa to induce apoptosis is exquisitely specific for proliferating endothelial cells, though HKa inhibits the spreading of a number of cell types, including osteosarcoma and melanoma cells, monocytes, and platelets (52) . These results raise the possibility that HKa might interact with a receptor selectively expressed on proliferating endothelial cells, perhaps inducing signaling responses that disrupt or circumvent common integrin signaling pathways required for maintenance of cell viability (53) . However, our studies do not suggest a role for any of the known endothelial cell HK or HKa receptors in mediating these effects, since antibodies that inhibit the binding of HK or HKa to these receptors do not affect the antiproliferative activity of HKa (Table 2) .

The effects of HKa demonstrate remarkable molecular specificity, since the two-chain molecule inhibited endothelial proliferation and induced apoptosis far more potently than HK or LK. Though our studies do not exclude the possibility that HK itself may inhibit endothelial cell proliferation, our data suggest that the small amount of HKa generated during the 48 h proliferation assay may be sufficient to account for the apparent activity of HK. Definitive resolution of this issue will require expression and functional testing of HK mutants resistant to kallikrein-mediated cleavage.

Our results support the paradigm in which conformational alteration of specific proteins, often belonging to the coagulation cascade (58) , is thought to lead to exposure of antiangiogenic neoepitopes (2 , 8) . The observation that cleavage of HK to HKa is accompanied by a conformational change characterized by increased exposure of domain 5 (38) suggests that the endothelial cell binding regions within this domain (21 , 30) may mediate the activity of HKa. This hypothesis is supported by our studies with recombinant domain 5 and domain 5-derived peptides (Table 3) , with peptides most active in inhibition of endothelial cell proliferation occurring within the previously described high-affinity endothelial binding region of this domain (30) . We speculate that the conformational change that occurs after HK cleavage allows this region to interact in a novel manner with endothelial cells, perhaps by binding to a receptor that does not recognize HK or does so with only low affinity. Indeed, like thrombospondin 1, which binds to at least 10 distinct cell surface molecules (59) , HKa may have yet undefined endothelial receptors that mediate these effects.

Several additional issues concerning the antiangiogenic properties of HKa warrant discussion. First, the dependence of the antiangiogenic activity of HKa on Zn2+ suggests potential overlap between the mechanisms of HKa and endostatin. Endostatin exists as a zinc-dependent dimer, which coordinates Zn2+ through interactions with three amino-terminal histidines and an aspartic acid (60) . The presence of Zn2+ within endostatin has been documented by physicochemical methods, and site-directed mutagenesis of the endostatin Zn2+ coordination ligands has confirmed their importance in its biological activity and/or stability (14) . HKa domain 5 contains at least two potential Zn2+ coordination sites, and the binding of HK to endothelial cells is Zn2+ dependent (45) . Taken together, these results suggest that HKa and endostatin might share a common Zn2+-dependent pharmacophore that mediates their antiangiogenic activity or stabilizes the active conformation of the molecules.

Colman et al. have recently reported that domain 5 of kininogen inhibits endothelial cell proliferation and migration, as well as angiogenesis in the chick chorioallantoic membrane (61) . These authors reported that a recombinant GST domain 5 fusion protein encompassing HK residues 420–513 inhibited endothelial cell proliferation by 50% at a concentration of ~50 nM, whereas a synthetic peptide corresponding to amino acids 440–455 of HK domain 5 inhibited proliferation to a similar extent at a concentration of ~110 nM (61) . Our studies extend these observations, as we have demonstrated that cleavage of naturally occurring single-chain high molecular weight kininogen to its two-chain form yields a potent antiangiogenic molecule, and that the activity of HKa, as well as that of domain 5 (Figs. 2 and 8) and the domain 5-derived peptides (not shown), is Zn2+ dependent. We have also demonstrated that HKa inhibits angiogenesis in mice. However, our studies do not support a role for the interaction between HKa or HK domain 5 and the urokinase receptor as contributing significantly to the antiproliferative activity of HKa, as proposed by Colman et al., since an antibody that blocks the binding of HKa to this receptor did not diminish the effects of HKa on endothelial cell proliferation. Moreover, we observed minimal inhibition of endothelial cell proliferation by the G440-H445 peptide, instead detecting much more potent activity in two peptides (H5–13, H5–14) from the carboxyl-terminal region of domain 5 previously reported to mediate high-affinity binding of HK to endothelial cells (Table 3) (30) .

Several considerations suggest that HKa may contribute to the physiological regulation of angiogenesis, particularly under inflammatory conditions. HK is a substrate for other proteases in addition to kallikrein, such as neutrophil elastase and mast cell tryptase, which in combination efficiently release bradykinin from HK (62) . Plasmin promotes the proteolysis of HK through direct cleavage between Lys420-His421 (one amino acid carboxyl-terminal to the kallikrein cleavage site) and Arg371-Ser372 (63) , as well as through activation of plasma kallikrein (63) . Though HK is the most abundant species of high molecular weight kininogen in normal human plasma, HKa and other kininogen degradation products predominate in pathological conditions such as disseminated intravascular coagulation (64) , trauma (64 , 65) , hereditary angioedema (66) , and thrombotic thrombocytopenic purpura (67) . Contact activation, accompanied by generation of HKa, also occurs in acute myocardial syndromes (68) and is stimulated by plasmin during pharmacological thrombolysis (69) . We are currently investigating the possibility that HKa and/or other kininogen-derived peptides circulate at increased levels in the plasma of patients with malignancy.

In summary, our studies demonstrate that HKa induces endothelial apoptosis and inhibits angiogenesis. Further characterization of the mechanism(s) by which HKa exerts these effects should provide new information concerning the physiological role(s) of this intriguing protein as well as insight into the regulation of angiogenesis.


   ACKNOWLEDGMENTS
 
We would like to acknowledge the contribution of Li Tan for assistance with DAPI staining, Bernd Binder (Technoclone, Austria) for his gift of human dermal microvascular endothelial cells, Berhane Ghebrehiwet for providing anti-gC1qR antibodies, and Alvin Schmaier for providing antibodies against the HK binding region of cytokeratin 1 as well as the PGG-15 peptide. This work was supported by National Institutes of Health grants HL50827, CA83134, and an Established Investigator Award from the American Heart Association (to K.R.M.). J.-C.Z. is the recipient of a postdoctoral fellowship (9920594V) from the Ohio Valley affiliate of the American Heart Association.

Received for publication December 8, 1999. Revision received June 2, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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