Download Potential treatment effect of Angiopoietin

Potential
treatment
neovascularization
effect
of
Angiopoietin-1
for
choroidal
1
Ying Wang1, Hongsheng Bi2,3, Da Teng1, Yingying Zou1, Xuemei Pan1, Dadong Guo3, Yan Cui2,3*
1Shandong University of Traditional Medinice,Yingxiongshan Road 48#, Jinan, China
2the Affiliated Eye Hospital of Shandong University of Traditional Chinese Medicine, Yingxiongshan
Road 48#,Jinan, China
3Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy
of Ocular Diseases in Universities of Shandong, Yingxiongshan Road 48#,Jinan , China
ABSTRACT
Objectives: To observe the effect of VEGF and Ang-1 on Norway Brown rat CNV model.
Methods: Our study was conducted at Eye Institute of Shandong University of Traditional
Chinese Medicine from June 2012 to June 2013. CNV model of rat were induced by laser. After
intravitreous injection of rVEGF or rAng-1, FFA and pathological exam were analyzed to detect
their effects. BRECs cultured in vitro MTT assay was applied to detect the proliferation ability
under hVEGF and hAng-1. The endothelial cell morphology, the changes of microcellular
structure and intercellular connection under hVEGF and hAng-1 were observed by TEM.
Results: In CNV rat model, less late leakage was found in Ang-1 group than control group and
VEGF group. MTT assay indicated VEGF could promote the proliferation of BRECs at low
concentration in dose-dependent manner. When VEGF concentration reached 50 ng/ml, the
promotion effect would switch to inhibition effect. When there is VEGF, the inhibition effect of
Ang-1 will not exist any longer. TEM of BRECs showed there are intercellular junction complex.
In VEGF group, there are more microvilli and less intercellular junction than control group. In the
VEGF + Ang-1 group, the intercellular junctions were nearly normal.
Conclusions: Although VEGF and Ang-1 are both important angiogenic factors,Ang-1 and VEGF
could accommodate each other’ when they coexisted. Ang-1 normalizes endothelial cell
ultrastructure and alleviates leakage of CNV. Ang-1 could be potential for CNV treatment.
Keywords
Vascular endothelial growth factor; Angiopoietin-1; Choroid neovascularization;
*Corresponence to:Yan Cui, Key Laboratory of Integrated Traditional Chinese and Western Medicine for
Prevention and Therapy of Ocular Diseases in Universities of Shandong, Affiliated Eye Hospital of Shandong
University of Traditional Chinese Medicine, Yingxiongshan Road 48#,Jinan, China. Phone:+86 531 58859638. Fax:
+86 531 82432074. [email protected]
Angiogenesis; Bovine retinal endothelial cells; Age-related macular degeneration
Introduction. CNV is frequently observed in patients with high myopia, angioid streaks,
inflammation, diabetic retinopathy and age-related macular degeneration (AMD). Although the
molecular basis for CNV is not well understood, an imbalance between pro-angiogenic and
antiangiogenic factors has been proposed in pathologic neovascularization. Vascular endothelial
growth factor (VEGF) has been identified as a validated target for several retinal vascular diseases,
including CNV.1-2 In turn, this led to the development of VEGF-neutralizing oligonucleotide
aptamer (pegaptanib),1 humanized anti-VEGF monoclonal antibody bevacizumab (Avastin)3 and
humanized anti-VEGF monoclonal antibody fragment (ranibizumab)4 for CNV treatment.
Antiangiogenic strategies could be particularly beneficial in preventing and treating the
progression of these diseases. But, it has been shown that retinal pigment epithelium
(RPE)-derived soluble VEGF has a role in maintenance of the choriocapillaris3 and that VEGF is
neuroprotective in the retina. Complete blocking of VEGF could alleviate neovascularization. It
could also block the positive effects of VEGF and bring some side effects such as systemic
thrombosis occurring5. Following the discovery of the VEGF-VEGFR system, the Tie receptors,
together with their corresponding angiopoietin (Ang) ligands, were identified as the second
EC-specific receptor Tye kinase signaling system.6 Angiopoietin-Tie system was defined by
different experiments the roles in controlling sprouting angiogenesis. In addition to Ang-1
activating Tie2 signaling, recent reports show that Ang-1 directly interacts with myocytes,
endothelial cells, and fibroblasts through integrins to mediate survival, cell adhesion, migration,
vascular remodeling and the transition from the quiescent to the activated EC phenotype.7 When
neovascularization is induced by VEGF under some pathophysiological circumstances,
angiopoietin remodels and matures the abnormal neovessles and alleviates leakage and
haemorrhage. That will be a new pathway for neovacularization treatment.
Materials and methods. Animals and reagents Pathogen-free female Brown Norway rats
(200-300 g) were purchased from Vital River Laboratories (Beijing) and were housed and
maintained in the animal facilities of the Eye Institute of Shandong University of TCM. All animal
studies conformed to the ARVO statement on the use of animals in Ophthalmic and Vision
Research. Institutional approval was obtained, and institutional guidelines regarding animal
experiment were followed. Ang-1 and VEGF of human and rat were purchased from R&D
systems (Minneapolis, MN). Our study was conducted at Eye Institute of Shandong University of
Traditional Chinese Medicine from June 2012 to June 2013.
Laser-induced choroidal neovascularization rat model and intravitreous cavity
injection Rats were anesthetized with intraperitoneal injection of 5 mg/kg ketamine. Pupils were
dilated by topical administration of 1% tropicamide. Ten laser spots concentric with the optic
nerve were placed avoiding the major retinal vessels in the right eye of each rat using a diode laser
(Carl Zeiss, Germany). The laser parameters such as spot size, laser power and exposure time
were 100 µm, 120 mw and 0.1 s, respectively. The Bruch’s membrane breakage was confirmed by
the end point ‘bubble formation’. Rats with intraocular hemorrhage on laser administration were
excluded from the study.
After the laser burn, 30 rats were divided into three groups randomly with 10 rats each, the
Ang-1 group, the VEGF group and the control group. For these three groups, 50 μl Ang-1(0.2
μg/ml), 50 μl VEGF (0.2 μg/ml) and the same volume of PBS were injected to intravitreous
cavity of right eye, respectively. The injections were performed on D1 and repeated on D3 after
laser surgery.
Fundus photography and fluorescein fundus angiography. Fundus photography and
fluorescein fundus angiography (FFA) were performed on three groups of rats after laser treatment
and intravitreal injection. FFA was performed on day 14. Rats were intraperitoneal injected with
0.5 mg/kg 10% fluorescein sodium. Photographs were taken by a fundus camera (Heidelberg,
Germany). Leakage of fluorescein associated with CNV lesions was monitored at several time
points until 30 minutes later after the dye injection including the arterial phase, early arteriovenous
phase and late arteriovenous phases. The FFA image output to UTSCSA image analysis software.
The background of the image of the entire FFA grayscale calibration 100 (± 1) grayscale.
Measured circular area which is the optic disc as the center 7.40 mm as the radius and the gray of
CNV leakage area in the three groups. Repeated three times, and take the average value.
Pathology. Neovascularization status in the eye was confirmed by histopathology. The rats
were sacrificed with overdose sodium pentobarbital and the whole eyes were collected 14 days
after laser treatment. The whole eyes were immersed for 1 h in 4% phosphate buffered
glutaraldehyde, and transferred to 10% phosphate buffered formaldehyde until procession. The
fixed and dehydrated tissue was embedded in methacrylate and 5 μm sections cut through the
pupillary-optic nerve plane and stained with hematoxylin and eosin. Presence or absence of
neovascularization was evaluated blindly by examining six sections cut at different levels for each
eye.
Bovine retinal endothelial cells culture. BRECs were obtained from were harvested from
bovine eyes, as described by Barber8 and the cells were resuspended in DMEM supplemented
with 10% (v/v) fetal bovine albumin, 100 U/ml penicillin G and 100 mg/ml streptomycin
sulphate and plated in 25-mm² culture flasks (NEST, USA) and then incubated at 37℃under 5%
CO2. The culture medium was changed every other day. The third passage was used in the
following experiments.
Endothelial cells proliferation assay-MTT assay. To evaluate the bioactivity of Ang-1 and
VEGF on endothelial cells in vitro, the proliferation of BRECs was taken as an index. The cell
concentration was accommodated to 1.0×105 cells/ml density by culture media. 200ul cell
suspension solution was adding to each well of 96-well plate. Incubate the plates in 37℃, 5% CO2
overnight. Ang-1 or VEGF was added with different concentration or the two combinations. After
24 hours incubation, the media was replaced MTT containing media (5 mg/ml) each well for
additional 4 hours. DMSO 150μl/well was added after abandon of the media. Absorbance was
measured at 490 nm with an automatic microplate reader (ie, OD).
Transmission electron microscopy. BRECs were grown on thermanox cover slips to
confluence. Cells were then fixed in 2.5% glutaraldehyde and washed in 0.1 M cacodylate buffer
for about 30 min. For TEM, the fixed samples were osmicated with 1% osmium tetroxide,
dehydrated in ascending ethanol concentrations (70–100% in 6 steps), and embedded in a 1/1
mixture of epoxy resin and ethanol for 2 h. After 2 h, fresh epoxy resin was added to the samples
and they were left overnight. Then, the resin was polymerized for 2 days at 60℃. Ultrathin
sections were cut and contrasted with 3% uranyl acetate and lead citrate. Samples were visualized
with a Zeiss EM 906.
Statistical Analysis. Data were expressed as the mean ± SD for the results from at least three
separate experiments. All data were analyzed with the SPSS 16.0 statistical package. A one-way
analysis of independent samples t-test for two-sample comparisons was used to compare the
means of two groups. P < 0.05 was considered statistically significant.
Results. Ang-1 could counteract the permeability–inducing effects of VEGF. For Brown
Norway rats, CNV could be induced by laser (Figure 1). The present of CNV could be identified
by FFA (Figure 2). For CNV, hyperfluorescence develops around the burn, which progresses to
late diffuse leakage with dye pooling in the serous detachment surrounding the burning area. From
fundus photography, there was fresh haemorrhage beside the laser burn sites in VEGF group
(Figure 1B). For Ang-1 group, there was no haemorrhage and no exudates surrounding the laser
burn (Figure 1, C & D). Among the 30 rat models, on D14 after laser burn, CNV occurrence rate
of control group, VEGF group and Ang-1 group was (65.53 ± 3.53)%, (68.78 ± 4.75)% and (32.33
± 3.47)%, respectively. Ang-1 group showed late mild leakage comparing with control and VEGF
groups (Figure 2, C & D).
Ang-1 could stabilize and normalize the laser-induced CNV. The CNV of three groups
was confirmed by pathology. Laser burn could induce choroidal neovascularization in Norway
Brown rat effectively. In control and VEGF groups, there were neovessels and haemorrhage at
choroid layer (Figure 3, A & B). On the contrary, in Ang-1 group, the neovessels existed at
choroid layer with ‘dry’ and ‘mature’ appearance (Figure 3, C).
VEGF and Ang-1 could affect the proliferation ability of endothelial cells. In vitro,
VEGF could promote the proliferation of BRECs at low concentration in dose-dependent manner.
But, when VEGF concentration reaches 50 ng/ml, the promotion effect will switch to inhibition
effect. The proliferation decreases to 38% of the original value (Figure 4, A). Ang-1 could inhibit
proliferation of BRECs in vitro with dosage-dependent feature (Figure 4, B). When there is VEGF,
the inhibition effect of Ang-1 will not exist any longer. The proliferation of BRECs remains at the
stable level though Ang-1 concentration increases to 40 ng/ml. In VEGF and Ang-1 with different
concentration ratio such as, 5:10, 5:20, 5:40 and 10:10, the BRECs proliferations are same as that
in 5 ng/ml VEGF (Figure 4, C).
Transmission electron microscopy. In normal endothelial cells (EC), there were
intercellular junction complex such as tight junctions, adherens junctions and gap junctions.
Double-layer membrane and crest of mitochondria were normal (Figure 5, A). In VEGF group,
there were more microvilli and less intercellular junction than control group (Figure 5, B). There
are bundle like myofilament and numerous mitochondria (Figure 5,C & D). In the VEGF + Ang-1
group, the intercellular junctions were nearly normal (Figure 5, E).
Discussion. Choroidal neovascularization, results from a series of pathological events
affecting the retinal pigment epithelium, Bruch’s membrane, and the choroid. For Brown Norway
rats, CNV could be induced in Bruch’s membrane rupture caused by laser blasting. The VEGF
family of proteins is the most important family of angiogenic factors that controls blood vessel
formation.9 Translational exploitation of the VEGF-VEGF receptor research has, in 15 years, led
to the clinical implementation of anti-angiogenesis as the development of a vision-enhancing
treatment for age-related macular degeneration. The use of human VEGF as a potential stimulant
in therapeutic angiogenesis has been widely demonstrated.10 However, there is still an uncertainty
as to whether the presence of human VEGF alone would suffice in the achievement of functional
and mature vessels lined with vascular smooth muscles or pericytes. There is evidence that
excessive human VEGF expression could bring about pathological and immature vessel formation
and enhanced vascular wall permeability, and could lead to angioma formation.11
During pathological ocular angiogenesis, leakage and haemarrhage is usually resulted from
the immature neovascularization. If some factors could promote maturation of neovascularization,
the newly formed vessels will not leak and bleed. As we know, in normal physiological process,
there are some factors that guard the formation and maturation of the blood vessels. A protein that
maintains stability after maturation of newly grown capillaries is Angiopoietin-1. For laser
induced CNV, Ang-1 group showed late mild leakage comparing with control and VEGF groups.
Pathology examination confirmed that in Ang-1 group, the neovessels existed at choroid layer
with ‘dry’ and ‘mature’ appearance. Ang-1 could stabilize and normalize the laser-induced
CNV,Ang1 suppresses VEGF-induced breakdown of the blood–retinal barrier, it is generally
accepted that Ang1-mediated activation of Tie2 promotes vascular stabilization and quiescence,12
Ang1 inhibits VEGF-mediated increases in endothelial permeability.13 Ang-1 could be produced
by pericytes. Its presence in mature capillaries improves continuity of the basal membranes and
the adherence of pericytes to endothelial cells. During angiogenesis it promotes capillary growth.
The transgenic overexpression of Ang-1 decreases vessel leakiness in response to
permeability-inducing inflammatory agents.13 Systemic delivery of Ang-1 into adult mice has
similar effects that counteract the permeability inducing effects of VEGF administration.14
The stimulatory effects of VEGF on ECs proliferation have been well reported in vitro and in
vivo.15 As our in vitro results showed, 5 ng/ml VEGF could promote the proliferation of ECs
significantly. Ang-1 could inhibit proliferation of BRECs in vitro with dose-dependent feature.
When there is VEGF, the inhibition effect of Ang-1 will be reversed. The proliferation of BRECs
remains at the stable level though Ang-1 concentration increases to 40 ng/ml. So, the effect of
Ang-1 on ECs proliferation is weak when there is VEGF under some disease conditions. In VEGF
and Ang-1 with different concentration ratio such as, 5:10 and 10:10, the BRECs proliferations are
same as that in 5 ng/ml VEGF. When there is 10 ng/ml Ang-1, promotion effect of VEGF on
BRECs proliferation could not be identified. So, VEGF and Ang-1 are two important angiogenic
factors. Additionally, they could accommodate the effects on ECs of each other.
As we know, CNV and vascular leakage are the major causes of visual loss in exudative
AMD. VEGF expression is increased in CNV membranes in human exudative AMD patients and
in animal models induced by laser treatment.16-18 In exudative AMD, ECs transform from the
quiescent to the activated phenotype. The effect of Ang-1 on EC is not on cell proliferation but on
cell permeability. Ang-1 could reduce the leakage of the vessle bloods through Rho GTPases and
the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway and change the balance between
them.19
The resting endothelial layer establishes adherens junction and tight junctions, which
regulates peri-endothelial permeability. In vitro culture, VEGF evoked proliferating and
differentiating ECs with ultrastructural changes features such as less intercellular junctions. There
are more microvilli and numerous mitochondria. There are bundle like myofilament. Under VEGF,
ECs are in an activated but not normal state. Ang-1-induced Tie2 signaling is directly involved in
limiting ECs permeability because Ang-1 could help ECs intercellular junctions recover normal.
Ang-1 is known to have important functions in recruiting pericyte and smooth muscle cells for
maturation and maintenance of the vascular system20 and it has been shown to enhance collateral
vessel formation in an animal model with myocardial and hindlimb ischemia. 20-21Ang-1
overexpression stimulates pericyte coverage, resulting in a vasculature that is more mature in
appearance. The resulting vasculature seems to be normal, and therefore the process of
Ang1-mediated vessel stabilization has been named normalization.22 In addition, recent data also
suggested that altered migration may also contribute to pericyte loss in DR and that this
mechanism is regulated by signaling via the Ang-1/Tie-2 pathway.23
Ang-1 not only modifies the ultra-structure of ECs, but also improves ECs function. Our
study has shown that angiopoietin-1, has the ability to stabilize and assist in the maturation of
blood vessels. Taken together with our results, Ang-1 normalizes endothelial cell ultrastructure
and alleviates leakage of CNV, which could be used for CNV treatment.
Acknowledgements
This study was Supported by Natural Science Funding of Shandong Province, China(Y2006C101) and State
Natural Science Funding of China(81100658).
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Figure legends
Figure 1. Representative fundus photographs indicate Ang-1 treatment significantly reduced the
CNV leakage and hemorrhage comparing with the VEGF group and control group. There is
hemorrhage around laser scar in control group (A) and VEGF group (B). For Ang-1 group, there
was no hemorrhage and exudates surrounding the laser burn (C,D).
Figure 2. Representative FFA indicates Ang-1 treatment significantly reduced the CNV leakage
comparing with the VEGF group and control group. There is late dye leakage around laser scar in
control group (A) and VEGF group (B). For Ang-1 group, there is no leakage surrounding the
laser burn (C,D). The quantity of intensity and area of fluorescein leakage in the different groups
was shown in the chart (E).
Figure 3. The CNV of three groups was confirmed by pathological exam. Laser burn could induce
choroidal neovascularization in Norway Brown rat effectively. In control group and the group with
intravitreous cavity VEGF injection, there were neovessels and haemorrhage at choroid layer
(A,B). On the contrary, in group with intravitreous cavity Ang-1 injection, the neovessels existed
at choroid layer with ‘dry’ and ‘mature’ appearance (C).
Figure 4. In vitro, VEGF could promote the proliferation of BRECs at low concentration in
dose-dependent manner. But, when VEGF concentration reaches 50 ng/ml, the promotion effect
will switch to inhibition effect. The proliferation decreases to 38% of the original value (A). Ang-1
could inhibit proliferation of BRECs in vitro with dose-dependent feature (B). When there is
VEGF, the inhibition effect of Ang-1 will not exist any longer. The proliferation of BRECs
remains at the stable level though Ang-1 concentration increases to 40 ng/ml. In VEGF and Ang-1
with different concentration ratio such as, 5:10, 5:20, 5:40 and 10:10, the BRECs proliferations are
same as that in 5 ng/ml VEGF (C). *Comparing with control group (*P<0.05,**P<0.01)
Figure 5. Transmission electron microscopy of BRECs. In normal EC, there are intercellular
junction complex such as tight junctions, adherens junctions and gap junctions (black arrows).
Double-layer membrane and crest of mitochondria were normal (A). In VEGF group, there are
more microvilli and less intercellular junction (white arrow) than control group (B). There are
numerous mitochondria (C). There are bundle like myofilament(star)(D). In VEGF+Ang-1 group,
the intercellular junctions were nearly normal (black arrows) (E).