Chorion Allantois Membrane (CAM) Assay

Transcription

1 Chorion Allantois Membrane (CAM) Assay Introduction: The CAM assay was based on Nguyen et al. (1994). It is a simple and commonly used test to assess the angiogenesis inhibiting or stimulating properties from various compounds. We ve evaluated the total polar extract of refined de-oiled jojoba flour and its major component i.e. the dimethylsimmondsin (44%) which was purified from this total polar extract. These tests were conducted at the Lab. of Experimental Cancerology (Ghent University, Belgium, Prof. Dr. M. Mareel & Dr. S. Van Bocxlaer). Material & methods: Fertilized chicken eggs are incubated during 4 days at 37 C. Subsequently, the egg shell is opened to disclose the CAM (Figure 1). Two to 3 ml of albumen is removed to reduce the pressure on the CAM in order to improve its manipulating properties. The opening is closed using cellophane tape and the eggs are further incubated until day 9 for application of the liquid test compounds using different concentrations. Dulbecco s modified Eagle medium (DMEM) was used as a negative control, whereas Vascular Endothelial Growth Factor (VEGF; a stimulator of angiogenesis) was used as a positive control. The total polar extract from refined, de-oiled jojoba flour that contains all biologic active components, dimethylsimmondsine purified from this extract and a commercially known angiogenesis inhibitor (Tangeretin) are compared in their angiogenesis inhibiting activities towards VEGF stimulated angiogenesis in the CAM s. Each solubilized test component is pipetted on a sterile plastic disc (10 mm diameter) and dried under sterile conditions. Each disc is treated with cortisone acetate (100 µg/50 µl) in order to avoid inflammatory reactions. After drying, the discs are placed on the CAM with the charged side facing the CAM (Figure 1). The control disc (DMEM) is placed at 1 cm of the disc containing the test component. The opening is sealed with cellophane tape and the eggs are further incubated. At day 11, the tape and discs are removed and 10% buffered formalin is sprayed over the CAM. Eggs are allowed to dry for 2 to 4 h at room T. Then, the CAM is cut out of the egg and mounted on a glass slide. The vascular density index is measured according to Harris-Hooker et al. (1983); all micro vessels that cross 3 concentric circles with a diameter of 6, 8 and 10 mm are counted (Figure 1).

2 The angiogenetic Index (AI) is expressed as {(t/c)x100}-100, whereby t = total number of crossing vessels where the disc containing the test compound was located, and c = total number of crossing vessels where the control disc was located. The test compound is classified positively (angiogenesis stimulating) when AI > 10% and negatively (angiogenesis inhibiting) when AI < 10%. When AI ranges between 10% and +10%, no effect is attributed to the examined test compound.

3 Results: All stock solutions were made in DMSO. DMEM was used as negative control in the tests and used to dilute the VEGF, test compounds and positive control compound (tangeretin) prior to application in the test. Total polar extract and dimethyl-simmondsin purified from this extract were applied in the same concentration (i.e. 5% in DMEM).

4 Experiment n 1 CAM Test : Experiment 1 Test Substance Max 16,13 7,92 61,04-7,92-7,46 12,84 0,08-12,26 Average -7,00-19,04 43,06-15,50-15,03-3,32-22,22-17,85 Min - 27,97-59,40 15,12-18,64-27,63-12,66-35,80-23,88 StDev 20,72 22,57 15,15 4,33 8,10 10,37 13,47 4,24 StError 9,27 9,22 5,73 1,94 3,62 4,64 5,50 1,89 # samples Exp 1 Units Max + StDev Average - StDev Min Mann - Whitney Statistical analysis Test substance ,0074 0,0045 0,0045 0,0045 0, , ,680-3,000 0, ,842 p-value 5-2, , , ,842 z-value Legend: 1 DMSO (Dimethylsulfoxide) 5 Total Polair extract + VEGF 2 DMEM 6 Dimethylsimmondsin 3 VEGF 7 Dimethylsimmondsin + VEGF 4 Total Polair extract 8 Tangeretin

5 Experiment n 2 CAM test : Experiment 2 Test Substance Max -16,70 112,70 20,00 1,11 Average -28,18 62,89-8,86-19,67 Min -35,00 31,80-33,30-35,70 StDev 6,94 30,52 19,59 15,09 StError 13,65 13,65 8,76 6,75 # samples Exp 2 Units Max + StDev Average - StDev Min Mann - Whitney Statistical analysis Test substance ,1172 0, ,0090 0,0090 p-value 3-1,567-2, ,940-2,611 z-value Legend: 1 DMEM 2 VEGF 3 Polair extract 4 Dimethylsimmondsin

6 Experiment n 3 CAM test : Experiment 3 Test Substance Max -6,56 47,47-14,56-11,72 Average -17,37 25,59-24,40-21,25 Min -25,00-11,27-33,33-27,18 StDev 6,71 22,44 7,70 6,54 StError 3,00 10,03 3,14 2,67 # samples Exp 3 Units Max + StDev Average - StDev Min Mann - Whitney Statistical analysis Test substance , ,402 0, ,739 0, ,739 z-value p- value Legend: 1 DMEM 2 VEGF 3 Total Polair extract + VEGF 4 Dimethylsimmondsin + VEGF

7 Conclusions: The VEGF stimulated CAM showed significant (p < 0.05) blood vessel growth compared with the negative control group (DMEM) in all 3 experiments. The difference between VEGF stimulated CAM and DMSO was also significantly different (p < 0.05) (Exp 1). Both the total jojoba extract and the pure dimethylsimmondsin have an inhibiting effect on angiogenesis which is not significantly different from the DMEM control group (See exp. 1 & 2) Both the total jojoba extract and the pure dimethylsimmondsin have an inhibiting effect on angiogenesis which is significantly different (p < 0.05) from the VEGF group (See exp. 1 & 2) The angiogenesis inhibitory effects of both the total polar extract as well as pure dimethylsimmondsin were comparable with tangeretin, a known angiogenesis inhibitor. (Exp. 1) Although pure dimethylsimmondsin was used in the same concentration as the total polar extract, its angiogenesis inhibiting properties were lower (but nevertheless still significant) than the total polar extract. This suggests that other products which are also present in the total polar extract but in much lower quantities than dimethylsimmondsin are more potent angiogenesis inhibiting agents. However, the possibility of a synergistic effect of the major and minor simmondsin molecules present in the total polar extract should be considered as well. Both the total jojoba extract and pure dimethylsimmondsin inhibit the angiogenetic effects of VEGF. The difference between VEGF and VEGF in combination with the total jojoba extract or dimethylsimmondsin is significant (see exp 1, 2 & 3). The addition of the used jojoba compounds, cancelled out the angiogenetic stimulating effect of VEGF completely, which suggests a real angiogenesis inhibiting effect of these compounds.

8 References: Harris-Hooker SA, Cajdusek CM, Wight TN and Schwartz SM (1983) Neovascular responses induced by cultured aortic endothelial cells. J. Cell Physiol. 114: Liekens S, Neyts J, Degrève B and De Clercq E (1997) The sulfonic acid polymers PAMPS and related analogues are highly potent inhibitors of angiogenesis. Oncology Res. 9: Nguyen M, Shing Y and Folkman J (1994) Quantitation of angiogenesis and anti-angiogenesis in the chick embryo chorioallantoic membrane. Microvasc. Res. 47(1):31-40