Angiogenesis, the growth of new
blood vessels, is a fundamental physiological process required for development,
reproduction, wound repair, and response to ischemia. Pathological
angiogenesis, often referred to as neo-vascularization, is associated with
disease conditions including retinopathies, arthritis, psoriasis and cancer
(Folkman, 1995). Since angiogenesis is a process that is generally down
regulated in healthy individuals, targeting of angiogenesis with safe
anti-angiogenic compounds, that are selective against newly formed vessels
while sparing existing ones, may not lead to side effects even after prolonged exposure.
The anticancer potential of berries has been related, at least in part, to a
multitude of bioactive phytochemicals that these colorful fruits contain.
Anticancer effects of berry bioactive are partially mediated through their abilities
to counteract, reduce, and also repair damage resulting from oxidative stress
and inflammation. In addition, berry bioactive also regulate carcinogen and
xenobiotic metabolizing enzymes, various transcription and growth factors,
inflammatory cytokines, and sub cellular signaling pathways of cancer cell
proliferation, apoptosis, and tumor angiogenesis (Seeram, 2008). Strawberries
are rich in anthocyanins which are member of flavonoid group of photochemical; chemically
anthrocyanins are glycosides and are water soluble in nature. Major
anthocyanins include cyanidin, pelargonidin, pentunidin. These are present in
strawberries and their juices as well.
Edible berry anthocyanins have been shown to inhibit cellular transformation
and it demonstrates their potent inhibitory effect on inducible VEGF
expression. Chicken chorioallantoic membrane (CAM)
is an extra embryonic membrane formed on day 4 of incubation. It has very thick
capillary network. Rapid capillary proliferation continues until day 11; the
mitotic index then declines just as rapidly, and the vascular system attains
its final arrangement on day 18, just before hatching (Ribatti et al. 2001). Chorioallantoic
membrane (CAM) assay is a valuable model for
evaluating angiogenesis and vasculogenesis and it has long been a favored
system for the study of tumor angiogenesis and metastasis (Ribatti et al.
2001). However, its utility has been limited due to difficulty in measuring the
angiogenic response to an experimental compound in an objective and quantifiable
manner. By utilizing a novel approach to quantify angiogenesis (Ejaz et al.
2004), we have adapted the CAM assay to create an in ovo angiogenesis model
system that is rigorously quantitative, amenable to high-throughput screening,
and applicable for the testing of systemic and/or topical administration of
experimental agents. The current experiment is designed to study the effects of
strawberry juice on angiogenesis by using chicken chorioallantoic membrane
assay (CAM assay).
Forty fresh fertilized eggs, of similar
weights were obtained from a local hatchery (Big Bird). All the eggs were
sprayed with 70% ethanol to reduce contamination from egg surface and air
dried. Eggs were then incubated at 37 ºC (humidity 55-60%) for 5 days. Fresh
strawberry fruits were immediately, without storage, squeezed to juice. The
edible portion of deep colored fruits were washed and chopped to squeeze fruit
juice using a manual stainless steel screw squeezer. Juice was centrifuged at
10,000 rpm (4 ºC) for 30 min. (Lin et al. 2008). The supernatant was then
collected and stored at -20 ºC for further use. On day 5 of incubation, eggs
were windowed aseptically as described by (Ejaz et al., 2005) with some
modifications. Briefly, a small window (approximately 2cm in diameter) was made
by removing the shell and inner shell membrane from the air-space site. On the
same day, 4 -5 ml of albumin was aspirated with a sterile syringe to allow the
embryos to develop in a way accessible to quantification. The windows were then
sealed with sterile Parailm
tape and eggs were returned to the incubator at 37 ºC (humidity 55-60%) and
incubated with the window upright till day 6 of incubation.
The 0.7% dilution was prepared using
distilled water. The pH of this dilution was then determined with the help of
pH meter and was adjusted in the range of 6.5-7.5. These dilutions were
filtered through Sartorius syringe filters (0.2 µm)
to reduce the risk of contamination. At day 6 of incubation windows were opened
and 200 µl of each dilution was applied on developing
CAM. Windows were sealed again with sterile Para-film
tape and eggs were kept in incubator for further 24 hours. Eggs were then
divided into two groups each having twenty eggs each. Group A was kept as
control which received distilled water only and group B received 0.7% dilution
of strawberry juice. Image probing system SPIP (scanning probe image
processor) was used for quantification of results. To ensure an objective 3D
measurement of angiogenesis on CAMs, serial images with respective x and y
dimensions were recorded, on day 7 of incubation, by Lebecca cam at 30 frames/s
using a camera shutter speed of 1/2000s. By using Adobe Photoshop 6.0 (Adobe
Systems Software- Ireland),
all of the images were converted into grayscale and contrast was improved by
black and white inversion, making every image possible to discern anatomical
structures and to facilitate precise quantification of angiogenesis (Ejaz et
After image acquisition, all images were
imported to scanning probe image processing software (IBM-Denmark) that works
on specific algorithm (Garnaes et al. 2006).
The diameter and length of various blood vessels was measured by using
calibration and measurement command. 3D surface roughness (fourteen parameters),
which is one of the main parameters in 3D image analysis, was also measured for
precise quantification of angiogenesis on the surface of CAMs. Vascular area,
abbot curve and angular spectrum of neovasculature on CAMs were calculated.
Thus blood vessels of micrometer and/or nanometer scale were quantified to
evaluate the in-depth effects of strawberry juice on angiogenesis (Ejaz et al,
All data was presented as mean ± SD.
Analysis of variance (ANOVA) was performed to evaluate different parameters
between controlled and treated samples; statistical significance was set at P
< 0.05. Post hoc Student’s t- test
was also performed when significance was found P < 0.05 (Melkonian et al.
RESULTS AND DISCUSSION
In this study we have examined the effect
of strawberry juice from fresh strawberry fruits on angiogenesis using chicken chorioallantoic
membrane (CAM) assay. We observed a
significant reduction in blood vessel formation by the application of single
dilution of strawberry juice with reference to control group. The following
results were obtained
In control samples, the blood vessels were
tremendously arranged in a tree like branching pattern with equivalent
distribution, covering the whole area of the CAM.
The vascular architecture of the CAM appeared
originating from the main “Y” branch of the blood vessel, which was further
differentiated into primary, secondary and tertiary branches (Fig. 1A)
Application of the 0.7% dilution of
strawberry juice caused marked changes in vascular architecture of the CAMs.
Anti-angiogenic activities were observed after application of dilution of
strawberry juice, which resulted in thinning of primary and secondary blood
vessels, and fading of tertiary blood vessels. This shows a marked reduction in
the complete vascular network of CAM (Fig.1B).
A novel image probing system (IPS) was
utilized for the assessment and quantification of structural changes in CAMs
caused by the application of dilution of strawberry juice.
SPIP was utilized for computerized
quantification of the diameter of CAM
vasculature. A significant reduction in diameter of primary, secondary and
tertiary blood vessels was evident among all treated groups as compared to
control group. A significant reduction in the diameter of primary, secondary and
tertiary blood vessels was recorded among strawberry juice treated groups (Fig.
Figure 1. Macroscopic evaluation of chicken Chorioallantoic membrane (CAM) at day 6 of incubation. Note the well defined
architecture of CAM blood vessels consisting of primary, secondary and tertiary
blood vessels in control group with well developed area of CAM (A), while CAM
treated with strawberry juice resulted in extensive decrease in CAM blood
vessels and reduction in total area of CAM representing extensive antiangiogenic
B = 0.7% dilution.
Diameter of the blood vessel on CAM of treated
Figure 3. Diameter of the blood vessel on CAM
of treated (0.7%) egg.
Table. 1. Roughness of control and treated CAMs
101.2 + 2.11 nm
70.32 + 3.250 nm
137.6 + 1.66 nm
101.3 + 3.55 nm
1.66 + 0.152
1.39 + 0.0085
1.966 + 0.137
2.67 + 0.099
255 + 1.300 nm
240 + 1.67 nm
255 + 1.300 nm
240 + 1.67 nm
1.296 + 0.014 nm
1.089 + 0.008 nm
0.00013 + 0.00006 nm
1.935 + 0.010
1.307 + 0.199
0.859 + 0.0028
359.7 + 5.65 nm
250.8 + 14.92 nm
0.70 + 0.0036
0.61 + 0.0076
Sa, average roughness; Sq,
root mean square deviation; Ssk, skewness of the surface; Sku, kurtosis of the
surface; Sdr, developed surface area ratio; Sci, core fluid retention; Sy,
lowest valley; Sz, maximum height of the surface; Ssc, arithmetic mean summit;
Sdq, root mean square slope; Spk, reduce summit height; Sti, texture index.
Fig. 4. Abbot curve of the blood vessels on CAM of control (A) and treated
(B) eggs showing less height of blood vessels on the CAM of treated sample (B)
than control (A).
For more accuracy, the 3D surface roughness
of normal and treated CAMs was measured. Parameters of 3D surface roughness of
CAMs were evaluated to quantify angiogenesis (Table. 1). The surface roughness,
representing neo-vascularization, of control CAMs was significantly greater
than that of treated CAMs. The average roughness values of control and treated
CAMs were 101.2 + 2.11 and 70.32 + 3.250 nm respectively.
Moreover, the Abbott curve, a graphic
representation of roughness, was also measured to evaluate even minute
differences in the height of blood vessels on the surface of CAMs. The heights of Abbott curves for control and
treated CAMs were 255 and 210 respectively (Fig. 4), showing that the height of
blood vessels on control cams was greater than that of treated CAMs. When ANOVA
is applied on all parameters, P-value was found to be than 0.05 (P < 0.05).
All the parameters evaluated demonstrate
the anti-angiogenic effect of strawberry juice in chicken Chorioallantoic
membrane. Our results showed that inhibition of angiogenesis by strawberry
juice may be due to suppression of endothelial cell spreading, migration and
angiogenesis. It is recommended that this area of research for strawberry juice
may be continued on tumor models to find out its efficacy against that
Faculty of Bio-sciences, Department of Pharmacology and
Toxicology, University of Veterinary and Animal Sciences, Lahore, Pakistan.
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