Marine cyclotripeptide X-13 promotes angiogenesis in zebrafish and human endothelial cells via PI3K/Akt/eNOS signaling pathways

Article abstract of DOI:10.3390/md10061307

Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently induced angiogenesis in zebrafish embryos and in human endothelial cells, which was accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of PI3K/Akt/eNOS by LY294002 or L-NAME suppressed X-13-induced angiogenesis. The present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.

Full text of DOI:10.3390/md10061307

Mar. Drugs 2012, 10, 1307-1320; doi:10.3390/md10061307
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Article
Marine Cyclotripeptide X-13 Promotes Angiogenesis in
Zebrafish and Human Endothelial Cells via PI3K/Akt/eNOS
Signaling Pathways
Xi-Lin Lu 1,†, Zhong-Liang Xu 2,†, Xiao-Li Yao 1, Feng-Juan Su 1, Cheng-Hui Ye 1, Jing Li 2,
Yong-Cheng Lin 2, Guang-Lei Wang 3,4, Jin-Sheng Zeng 1, Ru-Xun Huang 1, Jing-Song Ou 5,6
Hong-Shuo Sun 7, Li-Ping Wang 8, Ji-Yan Pang 2,* and Zhong Pei 1,*
,
1
Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080,
China; E-Mails: luxilin@mail.sysu.edu.cn (X.-L.L.); yeyaoxiaoli@sohu.com (X.-L.Y.);
jisufengjuan@163.com (F.-J.S.); ych201176@163.com (C.-H.Y.);
zengjs@pub.guangzhou.gd.cn (J.-S.Z.); hrx998@yahoo.com.cn (R.-X.H.)
School of Chemistry & Chemical Engineering, Sun Yat-sen University, Guangzhou 510275,
China; E-Mails: xzhliang1979@163.com (Z.-L.X.); zsulijing@163.com (J.L.);
ceslyc@mail.sysu.edu.cn (Y.-C.L.)
2
3
4
5
Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University,
Guangzhou 510080, China; E-Mail: wangglei@mail.sysu.edu.cn
Key Laboratory of Functional Molecules from Oceanic Microorganisms,
Department of Education of Guangdong Province, Guangzhou 510080, China
Division of Cardiac Surgery, The First Affiliated Hospital, Sun Yat-sen University,
Guangzhou 510080, China; E-Mail: oujs@mail.sysu.edu.cn
6
7
The Key Laboratory of Assisted Circulation, Ministry of Health, Guangzhou 510182, China
Division of Anatomy, Department of Surgery, Institute of Medical Science, Faculty of Medicine,
University of Toronto, Toronto, ON M5G 1G6, Canada; E-Mail: hss.sun@utoronto.ca
Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055,
China; E-Mail: lp.wang@siat.ac.cn
8
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: peizhong@yahoo.com (Z.P.);
cespjy@mail.sysu.edu.cn (J.-Y.P.); Tel.: +86-20-87755766 (ext. 8282) (Z.P.);
Fax: +86-20-87335935 (Z.P.); Tel.: +86-020-84036554 (J.-Y.P.); Fax: +86-020-84036554 (J.-Y.P.).
Received: 4 March 2012; in revised form: 8 May 2012 / Accepted: 29 May 2012 /
Published: 7 June 2012

Mar. Drugs 2012, 10
Abstract: Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A
1308
isolated from mangrove fungus Xylaria sp. (no. 2508). We found that X-13 dose-dependently
induced angiogenesis in zebrafish embryos and in human endothelial cells, which was
accompanied by increased phosphorylation of eNOS and Akt and NO release. Inhibition of
PI3K/Akt/eNOS by LY294002 or L-NAME suppressed X-13-induced angiogenesis. The
present work demonstrates that X-13 promotes angiogenesis via PI3K/Akt/eNOS pathways.
Keywords: angiogenesis; marine cyclotripeptide; zebrafish; endothelial nitric oxide
synthase; xyloallenoide A
Abbreviations
PI3K: phosphatidylinositol 3-kinase; Akt: alpha serine/threonine-protein kinase; eNOS: endothelial
nitric oxide synthase; HUVEC: human umbilical vein endothelial cells.
1. Introduction
Ischemic cardiac and cerebrovascular diseases are the leading causes of death and adult disability in
the world. In addition, many ischemic diseases such as limb ischemia also severely affect the daily life
of patients. Restoration of blood supply to the ischemic tissues and organs is the most critical aspect of
treatment of ischemic diseases.
Angiogenesis is the process of sprouting new blood vessels from existing blood vessels and plays a
pivotal role in reestablishment of blood flow to the ischemic organs to overcome the ischemic insult.
Thus, stimulation of angiogenesis represents an exciting approach for the treatment of ischemic
disorders and the clinical translation of novel angiogenic agents will benefit millions of patients
suffering from those disorders.
Zebrafish embryo is well suited to the study of blood vessel formation because the vascular system
has been well described in the developing zebrafish embryo and many pathways involved in
angiogenesis in mammals are highly conserved in the zebrafish. Recently, several transgenic zebrafish
lines with fluorescent blood vessels have been developed and these transgenic animals have been used
successfully in the field of angiogenesis drug screening [1]. Compounds can directly add to the fish
culture media, diffuse into the embryo and induce observable angiogenic effects.
The marine environment is a rich source of novel and unusual secondary metabolites for drug
discovery. Cyclotripeptide X-13 is a core of novel marine compound xyloallenoide A (Chart 1), which
was isolated from mangrove fungus Xylaria sp. (no. 2508) in the South China Sea [2]. Previously, we
have demonstrated the total synthesis of xyloallenoide A and its diastereomer xyloallenoide A1 [3]. To
explore their potential biological functions, we investigated whether these compounds have vascular
activity. In the present study, we first demonstrated that X-13 and its derivatives concentration-dependently
promoted the angiogenesis in zebrafish in vivo and endothelial cell cultures in vitro. The mechanism of
angiogenic action of X-13 was also investigated in endothelial cell cultures. X-13 induced phosphorylation
of eNOS and Akt in parallel with angiogenesis. Consistently, PI3K inhibitor or NOS inhibitor but not

Mar. Drugs 2012, 10
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ERK1/2 inhibitor significantly reduced X-13-induced phosphorylation of eNOS and Akt and
angiogenesis. Thus, our data suggest that X-13 induces angiogenesis through PI3K/Akt/eNOS pathway.
Chart 1. Structures of X-13 and Xyloallenoide A and its Derivatives.
2. Results and Discussion
2.1. The Synthesis of Marine Cyclotripeptide X-13 and Xyloallenoide A
All the tested compounds were synthesized according to the literature (Scheme 1) [3]. Generally,
Boc-
materials and BOP, EDCI, and BOP-Cl as coupling reagents. The N-methylation of Boc-
was performed with MeI/NaH in THF at room temperature, followed by esterification and deprotection
to form -MeVal-OMe (2). Dipeptide 3 was obtained by coupling 2 and Boc- -Lys(Cbz)-OH in 89%
yield, which was further subjected to saponification to provide 4 in 87% yield. The tripeptide 5 was
prepared by coupling dipeptide 4 with -MeAla-OMe or -MeAla-OMe in a good yield. The tripeptide
L
-Lys(Cbz)-OH, Boc-D-Val-OH,
L
-MeAla-OMe and
D-MeAla-OMe were used as starting
D-Val-OH (1)
D
L
L
D
5 was then saponificated using LiOH in THF/H2O, followed by removal of the Cbz group with
94% overall yield in two steps to afford the cyclizing precursor 7. The cyclpeptide 8 was smoothly
produced with EDCI/HOAt in moderate 56% yield. Final deprotection of the Boc group in 8 gave 9 in
96% yield. The xyloallenoide A and A1 was obtained by coupling of cyclictripeptide with
3-(4-buta-2,3-dienyloxy-phenyl)-acrylic acid [4] using BOP-Cl as catalyst in an acceptable 59% yield.
All the target molecules were purified through a flash column chromatography.
Scheme 1. Synthesis of X-13 and xyloallenoide A and derivatives.

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Scheme 1. Cont.
2.2. Angiogenic Activity in Zebrafish Angiogenesis Screen
Following synthesis of Cyclotripeptide X-13, their angiogenic action was examined in transgenic
zebrafish with fluorescent blood vessels (Figure 1). When zebrafish embryos were incubated with
tested compounds at final concentration 100 μM for 72 h, all compounds induced development of
ectopic subintestinal vessels (SIV) in zebrafish embryos as evidenced by longer and more numerous
spikes projecting from the subintestinal vessel basket, suggesting that all examined compounds
had angiogenic activity. The percentage of ectopic SIV was 7.8 ± 1.5% at control, 71.2 ± 5.9% at
X-13-treated group, 74.5 ± 6.4% at X-13a-treated group, 22.5 ± 3.6% at xyloallenoide A-treated group
and 28.3 ± 4.2% at xyloallenoide A1-treated group, respectively. Cyclotripeptide X-13 and X-13a had
stronger angiogenic action compared with nature marine product xyloallenoide A and synthesized
xyloallenoide A1. It should be noted that the steric hinerance of substituted allenic cinnamate of
xyloallenoide A and A1 at lysine residue was unfavorable for the activity. Furthermore, the difference
in angiogenic action was very subtle between compound X-13 and X-13a or between xyloallenoide A
and xyloallenoide A1, suggesting that absolute stereochemistry of alanine in the cyclotripeptide core is
not essential for their angiogenic action. Given that X-13 and X-13a had similar biological activity, the
nature core cyclotripeptide X-13 was used in the further bioactivity evaluation. In the dose response
experiments, cyclotripeptide X-13 at 10, 50 and 100 μM dose-dependently increased the number of
newly formed vessels and the plateau concentration was 50 μM (Figure 1). The angiogenic activity of
X-13 at 50 μM and beyond was comparable to that of VEGF at 100 ng/mL. Therefore, a dose of
50 μM X-13 was used for subsequent experiments.

Mar. Drugs 2012, 10
Figure 1. Effects of X-13 on angiogenesis in TG (Fli-1:EGFP) zebrafish embryos.
1311
(A) Representative brightfield images of zebrafish larvae from 48 to 96 hpf. Treatment
with X-13 for up to 96 h did not adversely affect the normal development of zebrafish
larvae; (B) Representative fluorescence microscopy images; and (C) The bar chart shows
quantitative data. X-13 (10, 50, 100 μM) dose-dependently induced angiogenesis in
zebrafish embryos with EGFP expressing SIV. The angiogenic activity of X-13 at 50 μM
and beyond was comparable to that of VEGF at 100 ng/mL. Data are expressed as the
mean ± SD. Results were obtained from six independent experiments (* p < 0.05 vs.
control vehicle group).
2.3. In Vitro Angiogenesis in Mammalian Endothelial Cells
Endothelial cells play a pivotal role in angiogenesis and activation of endothelial cells is the first
step in angiogenesis. To form new blood vessels, endothelial cells must go though basement membrane
invasion, cell migration, cell proliferation, and tube formation, of which every process can be a target
for intervention [5]. To investigate whether our zebrafish findings can be translated into human studies
and to explore the mechanisms responsible for X-13-induced angiogenesis, the biological action of

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X-13 was studied in Human Umbilical Vein Endothelial Cell (HUVEC) cultures. PI3K/Akt/eNOS and
ERK1/2 are two major pathways in endothelial angiogenesis. Therefore, the inhibitors for PI3K
(LY294002), eNOS (L-NAME) and ERK1/2 (PD98059) were used to explore the potential molecular
pathways underlying X-13-induced angiogenesis. We first examined the effect of X-13 on HUVEC
migration using the wound-healing method. There was no significant HUVECEC migration in vehicle
control-treated HUVECs at 12 h post-wounding. In contrast, X-13 treatment significantly increased
HUVEC migration (Figure 2A).
L-NAME or LY294002 but not PD98059 significantly inhibited
X-13-induced increase in HUVEC migration (Figure 2A). We then examined whether X-13 induces
endothelial cell invasion using transwell culture inserts. Compared with the vehicle controls, there was a
significant increase in the invasion of HUVECs treated with X-13. Treatment with L-NAME or LY294002
but not PD98059 significantly attenuated X-13-induced enhancement of HUVEC invasion (Figure 2B).
Figure 2. The effects of X-13 on Human Umbilical Vein Endothelial Cell (HUVEC)
invasion, migration and tube formation. HUVEC cultures were incubated with X-13 in the
presence or absence of inhibitors for different periods in different assays and VEGF-treated
cell cultures served as a positive control. Representative images show that X-13 induced
HUVEC migration (A), invasion (B) and tube formation (C) in HUVEC cultures. L-NAME
and LY294002 significantly inhibited X-13-induced cell invasion (A), migration (B) and
tube formation (C). The bar chart shows quantitative data. Data are expressed as the
mean ± SD. Results were obtained from four independent experiments (* X-13 vs. vehicle
control group, p < 0.05; # LY294002 plus X-13 vs. X-13, p < 0.05; ☆
vs. X-13, p < 0.05).
L-NAME plus X-13

Mar. Drugs 2012, 10
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Figure 2. Cont.
We further examined the effect of X-13 on HUVEC tube structure formation using Matrigel tube
formation assay. Treatment of HUVECs with X-13 induced an extensive formation of capillary-like
structures in a dose-dependent manner (Figure 2C). The increase in HUVEC tube formation was
comparable between X-13-treated (50 μM) and VEGF-treated (100 ng/mL) groups. Furthermore,
L
-NAME or LY294002 but not PD98059 inhibited X-13-induced HUVEC tube formation (Figure 2C).

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Consistent with our zebrafish findings, we found that X-13 treatment increased endothelial cell
invasion and augmented the migration of HUVECs, leading to a significant increase in HUVEC tube
formation, a hallmark feature of angiogenesis in endothelial cells.
2.4. PI3K/Akt/eNOS Activities in HUVECs
Understanding compound-induced signal pathway is essential for development of safe and effective
drugs. In previous experiments, L-NAME or LY294002 but not PD98059 inhibited X-13-induced
angiogenesis tube formation, suggesting that eNOS and PI3K pathways might be involved in
X-13-stimulated angiogenesis. To precisely investigate the signal transduction mechanisms responsible
for X-13-induced angiogenesis, we examined the role of PI3K/Akt/eNOS pathway in X-13-induced
angiogenesis. It is generally believed that endothelium-derived nitric oxide (NO) is a critical mediator
of angiogenesis. eNOS catalyzes the synthesis of NO in blood vessels and thus has an important role in
angiogenesis. It has been well documented that PI3K and its downstream effector Akt are implicated in
the activation of eNOS. When Akt is activated following PI3K stimulation, it phosphorylates eNOS
and the latter enhances NO release, thereby promoting angiogenesis [6]. In the present study, HUVECs
were treated with different concentrations of X-13 and phosphorylation and expression of eNOS and
Akt were examined by western blot (Figure 3). We found that X-13 dose-dependently increased Akt
phosphorylation at site 473 and eNOS phosphorylation at site 1177 without altering the expression of
total eNOS and Akt (Figure 3A,B). In contrast, X-13-induced Akt and eNOS phosphorylation was
abolished in the presence of PI3K inhibitor LY294002 (Figure 3C). In addition, X-13 significantly
increased NO generation while pre-incubation of HUVECs with L-NAME or LY294002 but not
PD98059 dramatically inhibited X-13-induced NO generation (Figure 3D). Together, these findings
suggest that PI3K/Akt/eNOS is responsible for the angiogenic action of X-13.
3. Experimental Section
3.1. Preparation of Reagents and Cell Culture
All reagents and solvents were of commercial quality and used without further purification. 1H and
13C NMR data were recorded on a Varian Inova 400 MB NMR spectrometer operating at 400 and
1
100 MHz for H and 13C respectively. All chemical shifts are in ppm (δ) with respect to
tetramethylsilane (TMS) as internal standard, and coupling constants (J) are in Hz. Mass Spectra on
DSQ (Low Resolution Mass Spectrometer) and MAT95XP (High Resolution Mass Spectrometer).
Melting point was determined on X-4 micro-melting point apparatus and was uncorrected. The purities
(>95%) of all target compounds were checked by HPLC using a LC-2010c equipped with UV detector.
Samples were injected on a Merck Purospher STAR RP-18e 125 cm × 4.6 mm (5 μm) column
equipped with a Merck Lichrocart precolumn (Merck, Darmstadt, Germany). HUVECs were obtained
from Sciencell, USA. HUVECs (Passage 4 to 7) were cultured at 37 °C in M199 media (Invitrogen,
Carlsbad, NM, USA) supplemented with 10% FBS. Cells were maintained in a humidified atmosphere
of 5% CO2. The angiogenic effect of X-13 on HUVECs was evaluated using a stock solution of X-13
(100 mg/mL) prepared in PBS containing 10% DMSO (GIBCO, Langley, VA, USA). The angiogenic
effect of X-13 on zebrafish was evaluated using a stock solution of X-13 (1 mg/mL) prepared in

Mar. Drugs 2012, 10
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sterilized Milli-Q water containing 0.5% DMSO. Vascular endothelial growth factor (VEGF) was
obtained from Sigma, St. Louis, MO, USA and prepared as a stock solution of 100 μg/mL in sterilized
Milli-Q water.
Figure 3. Activation of Akt/PI3K pathways and nitric oxide (NO) production in
X-13-treated HUVECs. Representative blots show that X-13 increased Akt and eNOS
phosphorylation (A,B) while PI3K inhibitor LY294002 attenuated X-13-induced Akt and
eNOS phosphorylation (C). Representative image shows that X-13 induced NO release
while PI3K inhibitor LY294002, NOS inhibitor LAME, but not erk1/2 inhibitor PD98059
reduced X-13-induced NO release (D). The bar charts show quantitative data. Data are
expressed as the mean ± SD. Results were obtained from three independent experiments
(* X-13 vs. vehicle control group, p < 0.05; # LY294002 plus X-13 vs. X-13, p < 0.05;
☆ L-NAME plus X-13 vs. X-13, p < 0.05).

Mar. Drugs 2012, 10
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Figure 3. Cont.

Mar. Drugs 2012, 10
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Figure 3. Cont.
3.2. Angiogenesis in Zebrafish Embryos
The transgenic zebrafish cell line TG (fli1: EGFP), in which endothelial cells express eGFP, was
kindly provided by ZFIN (Eugene, OR, USA) and maintained as described [7]. Zebrafish embryos
were generated by natural pair-wise mating of fish that were between 3 and 12 months old. Embryos
were collected as described. Healthy embryos were harvested at the 1–4 cell stage, plated in a 24-well
microplate and treated with different compounds. Embryos receiving DMSO (0.2%) served as a
vehicle control. VEGF, serving as positive control, was injected either into the yolk ball or into the
perivitelline space between the yolk and periderm. Embryos were incubated at 28 °C for 72 h and were
then anesthetized using 0.05% 2-phenoxyethanol in embryo water. One to three embryos were placed
in each well of a 96-well plate and pro-angiogenic effects were evaluated by examining each embryo
for the presence of ectopic vessels in the SIV as previously described [8]. The criteria for angiogenesis
includes: (a) presence of vessels spiking out of the basket structure; and/or (b) extension of the SIV
basket into the yolk extension region with more than seven vertical branches within the basket [8].
Thirty embryos in each experiment in each group were calculated.
3.3. Cell Invasion, Migration and Tube Formation
HUVEC invasion was investigated as described [9]. Briefly, the effect of X-13 on HUVEC invasion
was measured using the 10 mm tissue culture insert (transwell) with polycarbonate membrane
(8 mm pores) and 24-well companion plate. The upper side and lower side of the membrane were
pre-coated with 1:30 (v/v) and 1:100 (v/v) of Matrigel, respectively. The HUVECs were resuspended
in low serum (1% FBS) medium and seeded onto the culture inserts at 5 × 104 cells per insert in
triplicate. They were then deposited into the 24-well companion plate with 500 μL of low serum
(1% FBS) medium containing X-13 (50 μM) in the presence or absence of LY294002 (10 μM),

Mar. Drugs 2012, 10
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L
-NAME (100 μM) or PD98059 (50 μM). In addition, the wells of the companion plate, containing
DMSO (0.1%) and 100 ng/mL VEGF, served as a vehicle control and positive control, respectively.
The inserts were removed after 8 h of incubation and were then washed with PBS. Non-invasive cells
on the upper surface of the membrane were removed by wiping with cotton swabs. The inserts were
fixed in paraformaldehyate, stained with DAPI and mounted on microscope slides. Images of the
invasive cells were captured at 100× magnification using a fluorescent inverted microscope and a CCD
camera. Following this, HUVEC invasion was quantified by counting the number of cells per insert
with the software Metamorph Imaging Series (Molecular Devices, Tokyo, Japan).
HUVEC migration assay was performed using the wound healing method as previously described [10].
The HUVECs (3 × 105 cells) were seeded into each well of a 24-well plate and incubated with
complete medium at 37 °C and 5% CO2. After 24 h of incubation, cells were starved for additional
24 h by low serum (0.5% FBS) medium. The HUVECs were then scraped away horizontally in each
well using a P100 pipette tip. Three randomly selected views along the scraped line were photographed
on each well using a fluorescent inverted microscope and the CCD camera attached to the microscope
at 50× magnification. The medium was then changed to fresh low serum (1% FBS) medium with or
without X-13 (50 μM) in the presence or absence of LY294002 (10 μM), L-NAME (100 μM) or
PD98059 (50 μM). After 12 h of incubation, another set of images were taken by the same method.
Image analysis for signs of migration was performed by Metamorph Imaging Series. The average
scraped area of each well under each condition was measured and subtracted from that of the
before-treatment condition. Data are expressed as percentage wound closure relative to the wound
closure area in the control medium. The wound closure area of the control cells was set at 100%.
Endothelial tube formation was assessed in 24-well plates using growth factor-reduced Matrigel™
as described previously [11,12]. Briefly, growth factor-reduced Matrigel (250 μL) was pipetted onto
24-well culture plates and polymerized for 30 min at 37 °C. HUVECs were seeded on Matrigel-coated
plates at a density of 5 × 104 in low serum (1% FBS) medium containing X-13 together with or
without LY294002, L-NAME or PD98059 and then incubated at 37 °C for 8 h. Cells receiving DMSO
(0.1%) served as a vehicle control and were equivalent to no treatment. In addition, cells cultured in
100 ng/mL VEGF served as a positive control. The network-like structures were examined under an
inverted microscope (at 50× magnification). Tube-like structures were defined as endothelial cord
formations that were connected at both ends. The tube length was quantified using the software NIH
Image as reported earlier [11,12].
3.4. Western Immunoblot Analysis
HUVECs were plated in 60 mm dishes (16,700 cells/cm2) and cultured for 2 days. Culture medium
was then replaced with fresh medium containing 2% FBS and the cells were treated with different
concentrations of X-13 (10, 50, 100 μM) for 24 h. Cells were pretreated with or without LY294002
(10 μM) 30 min before exposure to X-13. Cellular proteins were harvested and immunoblots were
performed as described [13]. The primary antibodies used were: antibody to anti-eNOS (Santa Cruz
Biotechnology, Santa Cruz, CA, USA), anti-phosphorylation of eNOS at S1177 (P-eNOS S1177)
(Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (Cell Signaling Technology, USA),
anti-Akt (Cell Signaling Technology, USA), and anti-β-actin (Biosynthesis Biotechnology, Beijing, China).

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3.5. Measurement of NO Generation in HUVECs
NO generation was measured using a fluorescence method [14]. In this assay, 3-amino,
4-aminomethyl-2′,7′-difluorescein, diacetate (DAF-FM-DA; Beyotime, Shanghai, China) was used as
a fluorescent indicator of intracellular NO [14]. Briefly, HUVECs were seeded in 35-mm Petri dishes
with a glass-bottom insert (MatTek, Ashland, MA, USA) at the density of 4 × 105/well. After treatment
with X-13 (50 μM) for 1 h, Cells were washed three times with colorless serum-free medium (GIBCO,
Langley, VA, USA) and then incubated with 5 mmol/L DAF-FM-DA at 37 °C for 20 min. Cells were
then rinsed three times to remove the excess probe and maintained in colorless serum-free medium
throughout the experiments. DMSO (0.1%) and
L
-Arginine (1mM) were used as the vehicle control
-NAME (100 μM) or PD98059 (50 μM) were
and positive control, respectively. LY294002 (10 μM),
L
applied 10 min before X-13 treatment. Images were captured by an Olympus Fluoview laser confocal
scanning microscope (IX81; Olympus, Tokyo, Japan) with a FITC parameter (excitation 494 nm,
emission 518 nm). The fluorescence intensities were obtained using FV500 software (Olympus) to
quantify the image of each group.
3.6. Statistical Analysis
All data were presented as mean ± SEM. Differences among test groups were analyzed by ANOVA,
using Newman-Keuls multiple comparison test (Prism 4.0, GraphPad Software, Inc., San Diego, CA,
USA). A p value < 0.05 was considered statistically significant.
4. Conclusions
Hundreds of thousands of patients suffer from ischemic vascular diseases and the fundamental
problem underlying ischemic vascular diseases is the inability of tissue blood supply to match the
tissue demand. Thus, therapeutic angiogenesis has a great promise for those with ischemic vascular
diseases as therapeutic angiogenesis promotes angiogenesis and salvage ischemic tissues.
In the current study, we have identified Cyclotripeptide X-13 as a series of novel marine angiogenic
compounds. The PI3K/Akt/eNOS pathway is responsible for the angiogenic action of X-13 and the
exact mechanism of X-13 action on the PI3K/Akt/eNOS pathway is currently under investigation.
Given that X-13 is a small molecule with molecule weight under 1000 and possesses potent angiogenic
property, it is very promising for development as a novel class of pro-angiogenic agents for angiotherapy.
Acknowledgements
The authors thank the North Center of National zebrafish Resources of China for the technology
support in zebrafish. This study was supported by the grants from the National Natural Science
Foundation of China (No. 30770766, 30971261, 81041056, 20802094, 21172271), the National Key
New Drug Creation Program (2009ZX09103-039), the Science and Technology Planning Project of
Guangdong Province, China (No. 2008B080703021), Guangdong Natural Science Fund Committee
(No. 8151006001000007, 9251008901000003, S2011020001231), and the Fundamental Research
Funds for the Central Universities (11lgpy75).

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Samples Availability: Available from the authors.
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