The Indazole Derivative YD-3 Specifically Inhibits Thrombin-Induced Angiogenesis In Vitro and In Vivo

THE INDAZOLE DERIVATIVE YD-3 SPECIFICALLY INHIBITS

THROMBIN-INDUCED ANGIOGENESIS IN VITRO AND IN VIVO

Chieh-Yu Peng,* Shiow-Lin Pan,*

Hui-Chen Pai,* An-Chi Tsai,* Jih-Hwa Guh,

Ya-Ling Chang,* Sheng-Chu Kuo,

Fang-Yu Lee,

and Che-Ming Teng*

*Pharmacological Institute, College of Medicine, National Taiwan University;

Graduate Institute of

Pharmacology, Taipei Medical University; and

School of Pharmacy, College of Medicine, National Taiwan

University, Taipei; and

_{Graduate Institute of Pharmaceutical Chemistry, China Medical College;}

and

Yung-Shin Pharmaceutical Industry Co, Ltd, Taichung, Taiwan

Received 30 Oct 2009; first review completed 24 Nov 2009; accepted in final form 9 Mar 2010

ABSTRACT—Angiogenesis is a process that involves endothelial cell proliferation, migration, invasion, and tube formation, and the inhibition of these processes has implications for angiogenesis-mediated disorders. The purpose of this study was to examine the antiangiogenic efficacy of YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole], a selective thrombin inhibitor, on thrombin-induced endothelial cell proliferation and neoangiogenesis in a murine Matrigel model. First, the effect of YD-3 on angiogenesis was evaluated in vivo using the mouse Matrigel implant model. Plugs treated with 1 and 10_{2M of YD-3 inhibited neovascularization induced by thrombin, protease-activated receptor (PAR) 1, and PAR-4,} but not by vascular endothelial growth factor, in a concentration-dependent manner over 7 days. These results indicate that YD-3 has specific antiangiogenic activity on thrombin. YD-3 also inhibited (in a concentration-dependent manner) the ability of thrombin, PAR-1, and PAR-4, but not PAR-2, to induce the proliferation of human umbilical vascular endothelial cells, using a [3_{H]thymidine incorporation assay. YD-3 predominantly inhibited thrombin-induced vascular endothelial growth} factor receptor 2 (Flk-1) expression, but not extracellular signalYregulated kinase 1/2 phosphorylation, using Western blot analysis. YD-3 may have benefit in elucidating pathophysiology induced by thrombin-induced angiogenesis.

KEYWORDS—YD-3, thrombin, human umbilical vein endothelial cells, angiogenesis, Flk-1

ABBREVIATIONS—YD-3-[1-benzyl-3(ethoxycarbonylphenyl)-indazole]; PARs V protease-activated receptors; VEGF V vascular endothelial growth factor; ERK1/2 V extracellular signalYregulated kinase 1/2; HUVECs V human umbilical vein endothelial cells; ECGs V endothelial cell growth supplements; PKC V protein kinase C; SLIGKV V PAR-2-activating peptide (SER-LEU-ILE-GLY-LYS-VAL)

INTRODUCTION

Angiogenesis is the formation of new blood vessels from

preexisting endothelial vasculature (1). Physiologically,

angio-genesis plays a crucial role in embryonic development,

pla-cental implantation, and wound healing. In contrast, it supports

pathological conditions, such as solid tumor growth, diabetic

retinopathy, psoriasis, and rheumatoid arthritis. Complex and

diverse cellular actions, such as extracellular matrix

degrada-tion, proliferation and migration of endothelial cells, and

mor-phological differentiation of endothelial cells to form tubes,

have been implicated in angiogenesis (2). Although all these

processes are regulated under normal conditions, abnormal

vascularization is clearly implicated in tumor growth and

metastasis. The extreme growth of tumors to sizes larger than a

few cubic millimeters requires continuous recruitment of new

blood vessels (3). These newly synthesized blood vessels also

provide a route for cancer cells to enter the circulation and

spread to other, distant organs (4). Because of the importance

of angiogenesis, a simple and rapid

in vivo method to

deter-mine the antiangiogenic potential of compounds is desirable to

augment

in vitro findings, and the murine Matrigel-plug assay

has become the method of choice (5). Matrigel is extracted

from the Engelbreth-Holm-Swarm mouse sarcoma, a tumor

rich in extracellular matrix proteins. The major components of

Matrigel are laminin, collagen IV, heparin sulfate

proteogly-cans, entactin, and nidogen. Matrigel is mixed with angiogenic

factors, such as vascular endothelial growth factor (VEGF),

basic fibroblast growth factor, or IL-8 and injected

subcuta-neously into the ventral region of mice, where it solidifies,

forming a

BMatrigel plug.[ Endothelial cells migrate into the

plug and form vessels. Assessment of angiogenesis in the

Matrigel plug can be achieved either by measuring hemoglobin

or by scoring selected regions of histological sections for

vas-cular density (6).

Thrombin, a serine protease derived from the precursor

prothrombin, plays an important role in angiogenesis and is

a mediator of cellular effects that contribute to inflammation

reactions and the proliferation of endothelial cells in

tumori-genesis (7, 8). Many of the functions of thrombin are

medi-ated via activation of G protein

_{Ycoupled protease-activated}

receptors, PAR-1, PAR-3, or PAR-4 (9, 10).

Protease-activated receptors (PARs) are Protease-activated by an unusual,

irre-versible proteolytic mechanism in which the protease binds

to and cleaves the amino-terminal exodomain of the receptor.

This new amino terminus then binds intramolecularly to the

body of the receptor to initiate transmembrane signaling (11).

Recent studies have shown that thrombin has a significant

stimulatory effect on angiogenesis in that it can induce VEGF

Address reprint requests to Che-Ming Teng, PhD, Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd, Sect. 1, Taipei, Taiwan. E-mail: cmteng@ntu.edu.tw.

Chieh-Yu Peng and Shiow-Lin Pan contributed equally to this work. This study was supported by research grants from the National Science Council of the Republic of China (NSC 96-2628-B-002-109-MY3 and NSC 98-2321-B-002-022). DOI: 10.1097/SHK.0b013e3181df00a3

(12), VEGF receptor 2 (Flk-1) (13), angiopoietin 2 (14), and

!v"3 integrin (15) in endothelial cells.

In previous studies (16, 17), we showed that YD-3

[1-benzyl-3(ethoxycarbonylphenyl)-indazole], a new synthetic indazole

derivative, selectively inhibits rabbit platelet aggregation and

vascular smooth muscle cell proliferation caused by thrombin.

Alternatively, YD-3 selectively inhibits PAR-4

_Ydependent

platelet activation through blockade of PAR-4 and PAR-4

_Y

mediated thromboxane formation (18, 19). However, the role

of YD-3 in thrombin-induced angiogenesis is unclear. In this

study, we examined the ability of YD-3 to suppress

angiogene-sis

in vivo and in vitro and elucidated its mechanism of action.

Our data reveal that YD-3 specifically inhibits

thrombin-induced angiogenesis in a murine Matrigel model but does

not abolish the thrombin-induced angiogenic signal via

extra-cellular signal

Yregulated kinase (ERK), which critically

influ-ences cell proliferation in endothelial cells. In the present

study, we show that VEGF receptor 2 plays a predominant

role in thrombin-mediated angiogenesis. Together, these data

show that YD-3 inhibits thrombin-dependent endothelial cell

proliferation

in vitro and angiogenesis in vivo, by decreasing

Flk-1 expression. Further studies will be needed to characterize

the antiangiogenic effects of YD-3 more fully.

MATERIALS AND METHODS

The experimental protocol was approved by the Animal Care Committee of College of Medicine, National Taiwan University, and care and handling of the animals were performed in accordance with the National Institutes of Health guidelines.

In vivo matrigel plug assay

The murine Matrigel-plug assay can be used to evaluate antiangiogenic effect. Matrigel, an extract of mouse Engelbreth-Holm-Swarm tumor, is liquid at 4-C, and forms a gel when warmed to 37-C. It provides the essential substrates for the development of angiogenesis. Male BALB/c-nu mice (20 g, 4 weeks of age) were obtained from National Laboratory Animal Center, Taiwan, and acclimated to laboratory conditions 1 week before tumor implan-tation. BALB/c-nu mice were maintained in accordance with the Institutional Animal Care and Use Committee procedures and guidelines. Nude mice were given s.c. injections of 5002L of Matrigel (Becton Dickinson, Bedford, Mass) at 4-C with or without YD-3 (supplied by Yung-Shin Pharmaceutical Industry Co, Ltd, Taiwan) and thrombin, PAR-1Yactivating peptide (Ser-Phe-Leu-Leu-Arg-Asn, SFLLRN), PAR-4Yactivating peptide (Gly-Tyr-Pro-Gly-Lys-Phe, GYPGKF), and VEGF. After injection, the Matrigel rapidly formed a plug. After 7 days, animals were killed using an overdose injection of pentobar-bital (150 mg/kg); the skin of the mouse was easily pulled back to expose the Matrigel plug, which remained intact. After quantitative differences were noted and photographed, hemoglobin was measured, as an indication of blood vessel formation, using the Drabkin method (Drabkin reagent kit 525; Sigma, St Louis, Mo). The concentration of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel.

Cell culture

Human umbilical vein endothelial cells (HUVECs) were obtained from human umbilical cord veins with collagenase and cultured in 75-cm2 _plastic flasks in M199 containing 20% inactivated fetal bovine serum (FBS), 152g/mL endothelial cell growth supplements. Cells were incubated at 37-C in a humidi-fied atmosphere of 5% CO2in air. Media were changed every 2 days, and cells were passaged after treatment with a solution of 0.05% trypsin/0.02% EDTA. Experiments were conducted on HUVECs that had been used in passages 2 to 5.

[

H]thymidine incorporation assay

Confluent HUVECs were trypsinized, suspended in M199 supplemented with 20% FBS, and seeded at 5.0
103_{cells per well into 96-well plates. After} 24 h, the cells were starved with 2% FBS-M199 medium for 24 h. The cells were incubated with or without YD-3 and growth factors (thrombin, protease-activated receptors activating peptide, and VEGF) for 48 h and harvested. Before the harvest, cells were incubated with [3H]thymidine (22Ci/mL) for

16 h and harvested with Filter-Mate (Packard BioScience, Meriden, Conn), and incorporated radioactivity was determined.

Western blot analysis

After the exposure of cells to the indicated agents and time courses, cells were washed twice with ice-cold phosphate-buffered saline, and reaction was terminated by addition of 1002L ice-cold lysis buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 10 2g/mL aprotinin, 10 2g/mL leupeptin, and 1% Triton X-100). Protein (602g/lane) was separated by electrophoresis on a 5% to 10% sodium dode-cyl sulfateYpolyacrylamide gel electrophoresis. Proteins were electrophoreti-cally transferred to polyvinylidene difluoride membranes, and blots were blocked with 5% nonfat milk for 1 h. The membrane was immunoreacted with the primary antibody to ERK1/2, phosphorylated-ERK1/2 (BD Biosciences, Rockville, Md), and Flk-1 (Santa Cruz biotechnology, Inc, Santa Cruz, Calif) for overnight incubation at 4C. After four washings with phosphate-buffered saline/0.1% Tween 20, the secondary antibody (dilute 1:2,000) was applied to membranes for 1 h at room temperature. The antibody-reactive bands were performed with an enhanced chemiluminescence kit (ECL; Amersham Interna-tional, Little Chalfont, UK).

Data analysis and statistics

Data are presented as the meanT SE. Statistical significance was ensured by one-way ANOVA followed by the Tukey test for multiple comparisons. PG 0.05 was considered statistically significant.

FIG. 1. Effect of YD-3 on thrombin-induced neovascular formation in vivo. A, Antiangiogenesis effect of YD-3 in in vivo mouse Matrigel-plug assay. The experimental procedures are described under Materials and Methods. Matrigel without growth factors (thrombin 2 U/mL) did not show any migration or invasion of endothelial cells. However, with Matrigel containing growth factor, many blood vessels appeared in the gel on mice subcutaneous. Note the significant concentration-dependent inhibition of the formation of blood vessel in the gel after coplug of YD-3 for 7 days. B, Histological analysis (hematoxylin and eosin staining) of the effect of YD-3 on in vivo angio-genesis. Matrigel containing thrombin in vehicle-treated mice demonstrated a high degree of cellularity and the presence of blood-containing vessels (original magnification
100). C, Quantitation of active vasculature inside the Matrigel by measurement of hemoglobin content. Each value represents meanT SE (n = 5 or 6).#

P G 0.001 versus basal group; *P G 0.05 and ***PG 0.001 versus control group.

RESULTS

Effect of YD-3 on thrombin-induced neovascularization

in vivo

In a previous study, we showed that YD-3 specifically

in-hibited thrombin-induced vascular smooth muscle cell

prolif-eration (17). Thus, we decided to determine whether YD-3 was

capable of blocking thrombin-induced angiogenesis

in vivo.

Thrombin (2 U/mL) markedly increased the angiogenic

re-sponse, compared with Matrigel alone (Fig. 1, A and B), and

YD-3 (at 1 and 10

2M) significantly inhibited the angiogenic

response in a concentration-dependent manner. Microscopic

examination showed that the addition of thrombin to Matrigel

induced cellularity and induced the formation of cords, tubules,

and several blood-filled channels containing red blood cells.

In contrast, Matrigel pellets with no angiogenic agent had only

a few infiltrating, single, elongated cells. Thrombin-induced

angiogenesis was significantly reduced in mice treated with

thrombin plus YD-3, in a concentration-dependent manner.

Quantification of angiogenesis, using the hemoglobin content,

showed that the addition of thrombin to Matrigel induced an

angiogenic response, compared with Matrigel alone (Fig. 1C).

However, YD-3 also inhibited the thrombin-induced

hemoglo-bin content, in a concentration-dependent manner. These results

indicate that YD-3 is a potent antiangiogenic molecule

in vivo.

YD-3 selectively inhibits PAR-1Y and PAR-4Yinduced

neovascularization

in vivo

Thrombin stimulates cellular functions that are mediated

through the proteolytic activation of PAR-1 and PAR-4 (20).

Thus, we decided to determine the effects of YD-3 on the PAR-1

activating-peptide (AP)

_{Ymediated (100 2M) and PAR-4}

AP

_{Ymediated (500 2M) angiogenic functions. As shown in}

Figure 2, YD-3 (10

2M) significantly inhibited the PAR-1 APY

and PAR-4 AP

_{Yinduced angiogenic effects (Fig. 2). The}

thrombin-antagonizing action of YD-3 (100

2M) was verified

by its failure to inhibit angiogenesis stimulated by VEGF

(Fig. 3), a strong mitogen that induces angiogenesis. These

FIG_{. 2. Effect of YD-3 on PAR-1Y and PAR-4Yinduced neovascular} formation in vivo. Top, Antiangiogenesis effect of YD-3 in in vivo mouse Matrigel-plus assay. The experimental procedures are described under Materials and Methods. Matrigel without growth factors (1002M PAR-1 AP and 500_{2M PAR-4 AP) did not show any migration or invasion of endothelial} cells. Matrigel containing growth factor, many blood vessels appeared in the gel on mice subcutaneous. Note the significant inhibition of the formation of blood vessel in the gel after coplug of YD-3 for 7 days. Middle, Histological analysis (hematoxylin and eosin staining) of the effect of YD-3 on in vivo angiogenesis. Matrigel containing thrombin in vehicle-treated mice demon-strated a high degree of cellularity and the presence of blood-containing vessels (original magnification
100). Bottom, Quantitation of active vascu-lature inside the Matrigel by measurement of hemoglobin content. Each value represents meanT SE (n = 5 or 6).##_P

G 0.01 versus basal group; *P G 0.05 and **PG 0.01 versus PAR-1 APY and PAR-4 APYtreated group, respectively.

FIG. 3. Effect of YD-3 on VEGF-induced neovascular formation in vivo. Top, Antiangiogenesis effect of YD-3 in in vivo mouse Matrigel-plus assay. The experimental procedures are described under Materials and Methods. Matrigel without VEGF did not show any migration or invasion of endothelial cells. However, with Matrigel containing growth factor, many blood vessels appeared in the gel on mice subcutaneous. There is no significant inhibition of the formation of blood vessel between vehicle- and YD-3Ytreated groups. Middle, Histological analysis (hematoxylin and eosin staining) of the effect of YD-3 on in vivo angiogenesis. Matrigel containing VEGF in vehicle-treated mice demonstrated a high degree of cellularity and the presence of blood-containing vessels (original magnification
100). Bottom, Quantitation of active vasculature inside the Matrigel by measurement of hemoglobin content. Each value represents meanT SE (n = 5 or 6).##_{PG 0.01 versus basal group.}

results suggest that YD-3 is a specific thrombin inhibitor that

decreases angiogenesis

in vivo.

Effect of YD-3 on thrombin- and PAR-induced endothelial

cell proliferation

The effect of YD-3 on thrombin (2 U/mL) and

PAR-mediated HUVEC growth was assessed using [

H]thymidine

incorporation. As shown in Figure 4A, YD-3 significantly

in-hibited the thrombin-induced increase of DNA synthesis, in a

concentration-dependent manner (IC

= 1.1

10

j5

M). On

the other hand, YD-3 specifically suppressed cell proliferation

induced by PAR-1 AP (Fig. 4B) and PAR-4 AP (Fig. 4C) in a

concentration-dependent fashion (IC

= 1.1

10

M and

6.9

10

M, respectively), but did not affect the cell

pro-liferation induced by PAR-2

_{YAP (SLIGKV, Fig. 4D).}

Effect of YD-3 on ERK1/2 phosphorylation induced by

thrombin and PARs

It has been established that mitogen-activated protein

kinases (MAPKs), components in the signaling pathway, are

activated during the stimulation of cell proliferation (21).

Thus, we determined whether YD-3 inhibits thrombin- and

PAR-induced activation of ERK1/2 in HUVECs. As shown in

Figure 5, thrombin, PAR-1 AP, PAR-2 AP, and PAR-4 AP

induced a profound increase in ERK1/2 activation. YD-3 did

not suppress thrombin-induced ERK1/2 phosphorylation in

HUVECs (Fig. 5A). On the other hand, PD98059, a selective

MAPK inhibitor (it inhibits MEK), markedly inhibited the

effects of thrombin. No inhibition was observed with YD-3

on PAR-1 AP, PAR-4 AP, or PAR-2 AP. Moreover, trypsin,

a specific PAR-2 agonist, stimulated ERK1/2 activation

(Fig. 5, B and C), indicating that ERK1/2 does not plays a

major role in YD-3

_{Ymedicated inhibition of thrombin-induced}

endothelial cell proliferation.

Effect of YD-3 on thrombin-induced VEGF receptor 2

upregulation

The VEGF receptor, which drives endothelial cell

prolifera-tion, is also highly expressed in these cells. In a previous

study, thrombin was shown to mediate upregulation of the

VEGF receptor in endothelial cells (13). To investigate whether

FIG. 4. Effect of YD-3 on thrombin- and PAR-induced endothelial cell proliferation. Effects of YD-3 (1Y30 2M) on (A) thrombin (2 U/mL), (B) PAR-1 AP (100_{2M), (C) PAR-4 AP (500 2M), and (D) PAR-2 AP (100 2M) HUVEC} growth were examined using [3_{H]thymidine incorporation to assess} prolifera-tion. Data represent the meanT SEM of six independent experiments (each performed in triplicate).#PG 0.05,##

PG 0.01,###

PG 0.001 versus basal group; *PG 0.05, **P G 0.01, ***P G 0.001 versus control group. Without YD-3, only mitogen and vehicle-treated cells assigned as control group.

FIG. 5. Effect of YD-3 on ERK1/2 phosphorylation induced by thrombin and protease-activated receptors activating peptide. Human umbilical vein endothelial cells were incubated in the absence or presence of YD-3 for 1 h, and vehicle or angiogenic growth factors (A) thrombin (2 U/mL), (B) PAR-1 AP (1002M), 4 AP (500 2M), and (C) trypsin (0.3 nM), PAR-2 AP (100_{2M) were added to the cells for another 15 min. PD98059, a MEK} inhibitor, was used as positive control. Cells were harvested for the detection of phosphorylated-ERK1/2 and total ERK1/2 using Western blotting. Thrombin, PAR-1 AP, PAR-4 AP, PAR-2 AP, and trypsin induced a profound increase in ERK1/2 phosphorylation, and no significant inhibition was ob-served in YD-3Ytreated groups.

YD-3 inhibits thrombin-stimulated VEGF receptor

upregula-tion, HUVECs were stimulated with thrombin (2 U/mL), and

Flk-1 expression was evaluated by Western blot analysis.

Thrombin increased the expression of Flk-1 (Fig. 6), which was

involved in the mechanism of activation of angiogenesis by

thrombin. YD-3 significantly inhibited thrombin-induced Flk-1

upregulation in a concentration-dependent manner.

DISCUSSION

The purpose of this study was to examine the ability of

YD-3 to suppress angiogenesis

in vivo and in vitro and to

determine its specificity and mechanism of action. Our data

revealed that YD-3 significantly inhibited thrombin-induced

angiogenesis in a murine Matrigel model. In contrast, YD-3

had no or little inhibitory effect on the angiogenesis elicited

by VEGF, a highly angiogenic growth factor.

Angiogenesis, the process of new blood-vessel growth,

in-volves complex molecular signaling (22). Proliferation of

endothelial cells, a crucial event in angiogenesis, is regulated

by growth factors such as VEGF and basic fibroblast growth

factor (23). Thrombin also modulates endothelial cell

pro-liferation and angiogenesis (8, 24, 25).

YD-3, a low-molecular-weight nonpeptide thrombin

antago-nist, has an advantage over a direct thrombin inhibitor because

it does not inhibit the enzymatic action of thrombin in the

coagulation cascade, with minimal bleeding adverse effects.

Compared with peptide-mimic thrombin antagonists, YD-3

is also advantageous because the instability of peptides often

restricts their medical application. Moreover, YD-3 had good

oral availability in a previous study (17) and dual effects on both

PAR-1 and PAR-4.

It is well established that thrombin activates PAR-1, PAR-3,

and PAR-4 receptors (9). However, on the basis of studies with

vascular smooth muscle cells and platelets, it seems that YD-3

specifically blocks the action of PAR-1 and PAR-4 (17, 18). To

date, there is much functional evidence about 1 and

PAR-4 protein expression in endothelial cells (26, 27), and thus

PAR-1 and PAR-4 were considered to be the major thrombin

receptor in these cells. In this study, we demonstrated that the

addition of YD-3 significantly inhibited the proliferative effect

of thrombin, PAR-1 AP, and PAR-4 AP in HUVECs. These

results reveal that YD-3 acts via PAR-1 and PAR-4 to inhibit

thrombin-induced endothelial cell proliferation and then blocks

angiogenesis

in vivo.

Many studies have revealed that thrombin-induced

endo-thelial cell proliferation involves activation of protein kinase

C (PKC) (28, 29). Protein kinase C is found primarily in the

cytosol of unstimulated cells and becomes firmly associated

with the cell membrane after stimulation. In this study, we

found that YD-3 did not affect thrombin-stimulated PKC

translocation (data not shown). Additionally, ERK1/2 MAPK

is a key regulator of cell proliferation; it regulates gene

ex-pression and cell cycle reentry. Many growth factors and G

protein

_{Ycoupled receptor agonists induce cell proliferation via}

activation of ERK1/2 MAPK (30). Several lines of evidence

show that thrombin activates MAPK in a variety of cell types.

In the present study, we demonstrated that thrombin-induced

cell proliferation was mediated via activation of ERK1/2.

However, ERK1/2 phosphorylation induced by thrombin and

PAR peptides was not abolished by YD-3.

As previously noted, thrombin-induced angiogenesis is

asso-ciated with upregulation of VEGF (12) and the major VEGF

receptor, Flk-1 (13). Thrombin also upregulates

!v"3 integrin

(15) and matrix metalloproteinase 2 (31) in endothelial cells.

Recent evidence indicates a pivotal role for chemokine

growth-regulated oncogene

! in thrombin-induced angiogenesis (32).

All of these proteins contribute to thrombin-induced

angio-genesis. We demonstrated that thrombin-induced expressions

of VEGF and

!v"3 integrin were not affected by YD-3

treat-ment (data not shown). Moreover, YD-3 does not alter the

upregulation of chemokine growth-regulated oncogene

! and

activation of MMP-2 stimulated by thrombin (data not shown),

using reverse transcriptase

_{Ypolymerase chain reaction and}

zymography. In this study, compared with the basal group,

protein expression of the VEGF receptor (Flk-1) was

signifi-cantly increased at 24 h after thrombin stimulation and was

significantly inhibited in YD-3

_{Ytreated cells, suggesting that}

upregulation of the VEGF receptor may play a key role in the

thrombin-stimulated angiogenic response.

In conclusion, YD-3

Ymediated Flk-1 suppression may be

important for the inhibition of angiogenesis stimulated by

thrombin in endothelial cells. There are currently no effective

treatments for some angiogenesis-related diseases, such as

cancer, restenosis, and age-related macular degeneration.

Thrombin-induced angiogenesis may be involved in the

patho-logical process of these diseases. YD-3 used alone or in

com-bination with other agents may potentially be the treatment

for angiogenic disorders. Further investigation is required to

characterize the detailed molecular mechanism(s) and to

iden-tify the molecular target(s) associated with the antiangiogenic

activities of YD-3.

FIG. 6. Effect of YD-3 on thrombin-induced Flk-1 upregulation. Quiescent HUVECs were pretreated with dimethyl sulfoxide (CTL) or YD-3 (1, 10, 30_{2M) for 1 h and untreated (basal) or treated with thrombin (2 U/mL)} for another 24 h. Cell extracts were prepared, and equal amounts of protein were analyzed by sodium dodecyl sulfateYpolyacrylamide gel electropho-resis and immunoblotting with antibodies specific for Flk-1. The quantitative data are shown for ratio between basal and thrombin-treated groups. #

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