EFFECT OF DICLOFENAC SODIUM ANGIOGENESIS USING CHORIOALLANTOIC MEMBRANE (CAM) ASSAY
Iradat Hussain 1,2, Muhammad Ovais Omer2, Muhammad Ashraf2, Habib-Ur-Rehman2
1 Department of Pharmacy, Margalla Institute of Health Sciences, Islamabad, Pakistan 2 Faculty of Biosciences, University of Veterinary and Animal Sciences, Lahore, Pakistan
Keywords: Diclofenac sodium, Angiogenesis, Chorio-allantoic Membrane (CAM) assay.
Abstract

Angiogenesis, the formation of new blood vessels, is a hallmark of almost all neoplastic and non-neoplastic degenerative diseases. This process supports normal physiology as well as it contributes in progression of different diseases. Angiogenesis contributes in growth of tumor and progressive arthritis. Inflammatory mediators are involved in cancer induced angiogenic process. Cyclo-oxygenases promote these mediators which help in cell migration and endothelial cell spreading. To explore the role of diclofenac sodium in angiogenesis we have used in vitro Chorioallantoic membrane assay. A novel image probing system SPIP (scanning probe image processor) was utilized for assessment and quantification of structural changes in CAMs. Fourteen parameters of 3D surface roughness were also evaluated for quantification. Application of diclofenac sodium on Chorioallantoic membrane at day six of incubation (0.7% concentration of diclofenac sodium) showed anti-angiogenic effect. Results showed marked changes in architecture of CAMs, thinning of primary, secondary and tertiary blood vessels, reduction in surface roughness parameters, increase in kurtosis of surface, and decrease in Abbott curve. The substantial quantities of diclofenac sodium use locally may exhibit anti-angiogenic activity in the same manner those seen in in-vitro and explain its clinical efficacy.

Article Information

Identifiers and Pagination:
Year:2011
Volume:3
First Page:331
Last Page:345
Publisher Id:JAppPharm (2011 ). 3. 320-330
Article History:
Received:April 15, 2011
Accepted:June 27, 2011
Collection year:2011
First Published:July 20, 2011

INTRODUCTION
Angiogenesis, the growth of new capillary blood vessels in the body, has much more importance
in healing and reproduction. The body controls angiogenesis as there is a natural balance
between growth and inhibitory factors in healthy tissues. When this balance is disturbed, the
result is either too much or too little angiogenesis. Abnormal blood vessel growth cause serious
conditions like cancer, skin diseases, diabetic ulcers and many others.Folkman first hypothesized
in 1971 that solid tumors remain growth restricted to 2-3 mm in diameter until the onset of
angiogenesis (Folkman, 1971). Tumors derive blood supply from adjacent tissues, are an
important step in tumor growth, and are now well documented (Folkman, 2001). Inflammation
can stimulate angiogenesis and angiogenesis can facilitate inflammation. The sensitization of
sensory nerves by inflammatory mediators is also a source of pain, and sensitized nerves can
cause neurogenic inflammation and initiate angiogenesis (Bonnet and Walsh, 2004).inhibition of
angiogenesis caused by NSAIDs is a contributing factor in ulcer healing, a mechanism by which
NSAIDs inhibit angiogenesis is appear to be multifactor and includes local changes in
angiogenic growth factor expression, alteration in key regulators and mediators of vascular
endothelial growth factor ,increased endothelial cell apoptosis, inhibition of cell migration,
recruitment of inflammatory cells and platelets ( Klagsbrun , 1991).Function of chicken
Chorioallantoic membrane is supported by presence of dense capillary network. CAM has been
broadly used to study the morpho-functional aspects of the angiogenesis process in vivo due to
its extensive vascularization.Tumors remain avascular for 72 h, after which they are penetrated
by new blood vessels and begin a phase of rapid growth. The CAM may also used to verify the
ability to inhibit the growth of capillaries by implanting tumors onto the CAM and by comparing
tumor growth and vascularization with or without the administration of an anti-angiogenic
molecule.
Other studies using the tumor cells/CAM model have focused on the invasion of the chorionic
epithelium and the blood vessels by tumor cells. The cells invade the epithelium and the
mesenchymal connective tissue below, where they are found in the form of a dense bed of blood
vessels, which is a target for intravasation (Ribatti and Domenico, 2010).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). By utilizing a novel approach to quantify angiogenesis (Ejaz et al. 2004), we have
adapted the CAM assay to create an in vivo 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. Here we report on the effect of 0.7%
concentration of diclofenac sodium on angiogenesis using chicken Chorioallantoic membrane
(CAM) assay.


MATERIALS AND METHODS
Laid fertilized broiler chicken eggs obtained from a local hatchery were incubated at 37oC and
relative humidity of 55-60%. On day five of incubation, eggs were windowed aseptically as
described by (Ejaz et al., 2005). Briefly a small window (approximately 2cm in diameter) was
made by removing the shell and inner shell membrane. About 4-5 ml of albumin was removed,
windows were then sealed with Para film and eggs were returned to incubator.
0.7% concentration of diclofenac sodium was prepared using distilled water. The pH of the
dilution was then checked with the help of pH meter and was adjusted in the range of 6.5-7.5.
This dilution was filtered through syringe filter (0.2 µm) to reduce the risk of contamination.
Twenty chicken Chorioallantoic membranes (CAMs) of day six were used fore the present study.
Eggs were divided in two groups A and B containing ten eggs in each. On day six of incubation,
Group A received distilled water and kept as control while group B received 0.7% concentration
of diclofenac sodium. Windows were sealed again with sterile Para-film tape and eggs were kept
in incubator for further 24 hours.
Serial images of control and treated CAMs were recorded 24 hours after the administration of
diclofenac sodium (on day seven of incubation). The contrast between blood vessels and other
tissues was adjusted by using Adobe Photoshop 6.0, making it possible to discern the anatomical
structures on every image. These images were then imported to SPIP software (IBM Denmark),
an image processing program that works on specific algorithm (Garnaes et al., 2006), for
automatic measurement of surface roughness and related parameters for detailed evaluation of
the anti-angiogenic response.
The collected data from the study was analyzed by appropriate statistical procedure. 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 < 0.0 (Melkonian et al. 2002b).
RESULTS AND DISCUSSION
Application of the 0.7% concentration of diclofenac sodium caused marked changes in vascular
architecture of the CAMs. Anti-angiogenic activities were observed after application of 0.7%
concentration of diclofenac sodium, 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.1).
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 (Fig. 2).

Figure 1. Macroscopic evaluation of chicken chorio-allantoic membrane 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 diclofenac sodium resulted in extensive decrease in CAM
blood vessels and reduction in total area of CAM representing extensive anti-angiogenic
activities.
Figure 3. Abbott 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).
Table 1. Roughness of control and treated CAMs
For more accuracy, the 3D surface roughness of control and treated CAMs was measured. The
average roughness values of control were more as compared to treated CAM. This shows that
surface roughness, representing neo-vascularization, of treated CAMs was significantly (P <
0.05) less than that of control CAMs. Twelve parameters of surface roughness of CAMs were
calculated to quantify angiogenesis (Table.1). These parameters explain the differences in
surface roughness between control and treated CAMs. The Abbott curve, a graphical
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 the Abbott curve for control and treated
CAMs were 213 nm and 164 nm respectively (Fig.3).

CONCLUSION:
All the parameters evaluated demonstrate the anti-angiogenic effect diclofenac sodium in
chicken Chorioallantoic membrane. Our results showed that inhibition of angiogenesis by
diclofenac sodium may be due to suppression of alphaVbeta3 integrin mediated and Cdc42/ Racdependent
endothelial cell spreading, migration and angiogenesis. It is recommended that this
area of research for diclofenac sodium should continue to be explored, as this can lay the
foundation for the development of strategies for the prevention and therapy of several types of
angiogenesis dependent diseases.

ACKNOWLEDGEMENT:
1 Department of Pharmacy, Margalla Institute of Health Sciences, Islamabad, Pakistan
2 Faculty of Biosciences, University of Veterinary and Animal Sciences, Lahore, Pakistan
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Welcome to the research group of Prof. Dr. Cornelia M. Keck in Marburg. Cornelia M. Keck is a pharmacist and obtained her PhD in 2006 from the Freie Universität (FU) in Berlin. In 2009 she was appointed as Adjunct Professor for Pharmaceutical and Nutritional Nanotechnology at the University Putra Malaysia (UPM) and in 2011 she obtained her Venia legendi (Habilitation) at the Freie Universität Berlin and was appointed as a Professor for Pharmacology and Pharmaceutics at the University of Applied Sciences Kaiserslautern. Since 2016 she is Professor of Pharmaceutics and Biopharmaceutics at the Philipps-Universität Marburg. Her field of research is the development and characterization of innovative nanocarriers for improved delivery of poorly soluble actives for healthcare and cosmetics. Prof. Keck is executive board member of the German Association of Nanotechnology (Deutscher Verband Nanotechnologie), Vize-chairman of the unit „Dermocosmetics“ at the German Society of Dermopharmacy, active member in many pharmaceutical societies and member of the BfR Committee for Cosmetics at the Federal Institute for Risk Assessment (BfR).

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