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7.: Proof of principal: Progress Report 1
EFFECT OF SIMMONDSIN DERIVATES IN DIFFERENT ANGIOGENESIS ASSAYS
Dept. Vascular & Connective Tissue Research
TNO Prevention and Health
PO Box 2215
2301 CE LEIDEN
The Netherlands
Drafted by: Dr. P. Koolwijk & Dr. P. Quax
Sponsor of the study: D’Oosterlynck R&D
Project: D’Oosterlynck R&D
Study/project number: 011.30527
Date: January 15, 2008
CONTENTS
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Objective of the study
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Sponsor of the study
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Testing facility
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Responsible personnel
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Characterization of test substance
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Characterizations of the test system
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Observations, analyses and measurements
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Results
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Conclusions
1. Objective of the study
The aim of the study is to evaluate the effect of simmondsin derivatives in in vitro (and in vivo) angiogenesis.
2. Sponsor of the study
Name: D’Oosterlynck R&D
Address: D’Oosterlynck R&D
Museumlaan 17
9831 Sint-Martens-Latem, Belgium
Study Monitor: Mr. André d’Oosterlynck
E-mail: info@jojoba.be
3. Testing facility
Div. Vascular & Connective Tissue Research
TNO Prevention and Health
Zernikedreef 9
2333 CK LEIDEN
The Netherlands
4. Responsible personnel
Study director/monitor Dr. P. Koolwijk & Dr. P. Quax
Technical assistance Dr. E. Kaijzel, Ing. E. Peters
5. Characterization of test substance
The test substances were provided by D’Oosterlynck R&D as freeze-dried substances;
– compound A1: Total polar extract
– compound A2: simmondsins (partial purified, contains mainly
dimethylsimmondsin, desmethylsimmondsin (2 isomers) and
didesmethylsimmondsin, all in the hydroxylform)
– compound A3: simmondsin ferulates (mixture of ferulates of all in A2 described
simmondsins in the hydroxylform, +/- 65% pure)
– compound A4: dimethylsimmondsin (pure, in the hydroxylform)
– compound A5: dimethylsimmondsin ferulate (pure, in the hydroxylform)
– compound B3: 4-desmethylsimmondsin (CD671)
– compound B4: 5-desmethylsimmondsin (CD673)
– compound B5: didesmethylsimmondsin (CD910, pure in the hydroxylform)
– compound B6: partial purified simmondsin ferulates (mixture of all in A3 described simmondsin ferulates in the hydroxylform, +/- 87% pure)
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Stored under the conditions: 4°C.
The test substance were tested as specified in paragraph 6 + 7.
6. Characterization of the test systems
Incorporation of 3H-thymidine
Confluent cultures of HUVEC were detached by trypsin/EDTA solution, and allowed to adhere and spread at an appropriate cell density on gelatin-coated dishes in M199-HEPES medium supplemented with 10% heat-inactivated new born calve serum (NBCS) and penicillin/streptomycin. After 18 h the HUVEC were stimulated with 6.25 ng/ml vascular endothelial growth factor type A (VEGF-A) in M199-HEPES, penicillin/streptomycin, 10% NBCS in duplicate wells, with or without the indicated simmondsin derivatives. After an incubation period of 48 h, a tracer amount (0.5 µCi/well) of [3H]-thymidine was added and the cells are incubated for another 6 h period. Subsequently, the cells were washed with PBS, [3H]-labeled DNA was fixed with methanol, and precipitated in 5% trichloroacetic-acid, and finally dissolved in 0.5 ml 0.3 M NaOH and counted in a liquid scintillation counter.
In vitro angiogenesis assay
Human fibrin matrices were prepared by the addition of 0.1 U/ml thrombin to a commercially obtained (Chromogenix AB, Mölndal, Sweden) mixture of 2 mg/ml fibrinogen (final concentrations), 2 mg/ml Na-citrate, 0.8 mg/ml NaCl, 3 µg/ml plasminogen in M199 medium and 2.5 U/ml factor XIII. 100 µl aliquots of this mixture were added to the wells of 96-well plates. After clotting at room temperature, the fibrin matrices were soaked with M199 supplemented with 10% HS and 10% NBCS for 2 h at 37 oC to inactivate the thrombin. Frozen human microvascular endothelial cells (hMVEC, 0.7 x 105 cells/cm2) were thawed and seeded in a 1.8:1 split ratio on the fibrin matrices and cultured for 24 h in M199 medium supplemented with 10 % human serum, 10% NBCS, and penicillin/streptomycin. Then, the hMVEC were stimulated with the mediators and/or simmondsin derivatives for 7 days. Fresh medium, containing the mediators and/or inhibitors, were added every second day. Invading cells and the formation of tubular structures of hMVEC in the three-dimensional fibrin matrix were analyzed by phase contrast microscopy. The total length of tube-like structures of four microscopic fields (7.3 mm2/field) were (clockwise) measured using an Olympus CK2 microscope equipped with a monochrome CCD camera (MX5) connected to a computer with Optimas image analysis software, and expressed as mm/cm2 (Koolwijk et al. J. Cell Biol. 1996, 132:1177-1188).
7. Observations, analyses and measurements
All outcome measures were measured in singular, i.e. one measurement per culture well. The proliferation of HUVEC was expressed as mean ± range [3<H]-thymidine incorporation (dpm) of duplicate wells.
The percentage of inhibition of FGF-2-induced HUVEC proliferation by the simmondsin derivatives was calculated as follows:
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The data of the independent experiments were used to determine the average inhibition of the simmondsin derivatives.
[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/2″][vc_single_image image=”1565″ img_size=”full”][vc_column_text]Figure 1. Schematic representation of the in vitro angiogenesis model. Human foreskin MVEC are cultured on top of a 3-dimensional fibrin matrix and stimulated with a combination of a growth factor (e.g. bFGF or VEGF) and the inflammatory mediator TNFα. After some days the MVEC start to invade the fibrin matrix and form capillary-like tubular structures. Seven days after the first stimulation, and followed by 2 booster stimulations, the tube length is determined by image analysis (see Fig. 2).[/vc_column_text][/vc_column_inner][/vc_row_inner][vc_row_inner css=”.vc_custom_1530785286068{padding-bottom: 40px !important;}”][vc_column_inner][vc_single_image image=”1564″ img_size=”full”][vc_column_text]
Figure 2. Determination of the tube length by image analysis. A non-phase contrast microscopy image (A) is analyzed using dark field microscopy (B) and a video camera connected to the microscope. The tube-like structures are lightened up and stored in a computer. Then the image is skeletonized by the image analysis program Optimas™ and the total length of the structures of 4 microscopic fields is determined and expressed as mm/cm2.
8. Results
Angiogenesis, the formation of new microvessels from existing ones, occurs in various pathological conditions, such as tumors, rheumatoid arthritis and diabetic retinopathy. During the progress of angiogenesis, dynamic mutual interactions between endothelial cells and the extracellular matrix (ECM) take place. Inhibition of angiogenesis may be of therapeutic option to reduce/stop the growth of primary tumors and their metastases.
The regulation of angiogenesis is the result of a delicate balance between stimulators and inhibitors and involves several steps. After stimulation of the endothelial cells, the inner cell-layer of the blood vessels, by angiogenic factors, the basement membrane is degraded by proteolytic enzymes, in particular matrix-degrading metalloproteinases (MMPs) and enzymes of the plasminogen activator system. The cells will then invade, migrate and proliferate under the influence of angiogenic factors into the underlying interstitial matrix and will form new capillary structures. The D’Oosterlynck R&D compounds were tested on their capacity to inhibit endothelial cells proliferation and the formation of capillary-like tubular structures of endothelial cells in a 3-dimensional fibrin matrix.
A. Endothelial cell proliferation.
HUVEC proliferation was determined by measurement of the incorporation of 3H-thymidine incorporation after stimulation with the angiogenic growth factor VEGF. Two individual experiments were performed with different HUVEC isolations. High concentrations (between 2-5% (w/v)) of the tested compounds induced a general HUVEC cell death, as observed by the detachment of the cells from the culture plates (data not shown) and the negligible amount of 3H-thymidine incorporation (data not shown). Lower concentrations didn’t induced this cell death but inhibited the VEGF-induced HUVEC proliferation concentration dependent (see Figure 3). Compound A4 didn’t have any effect on the VEGF-induced HUVEC proliferation.
B. In vitro angiogenesis by hMVEC.
The ability of the D’Oosterlynck R&D compounds to inhibit in vitro angiogenesis was tested in a model in which human foreskin MVEC are cultured on top of a 3-dimensional fibrin matrix. After stimulation with a combination of a growth factor (in this case VEGF) and the inflammatory mediator TNFα, these HMVEC will start to invade the fibrin matrix and form capillary-like tubular structures. Seven days after the first stimulation, and followed by 2 booster stimulations, the tube length is determined by image analysis (see Fig. 2). Several experiments with different compounds were performed (see Figures 4 + 5). Also in these in vitro angiogenesis experiments it was observed that at high concentrations (2-5% (w/v)) cell death occurred (data not shown). After a cultured period of 7 days, all the hMVEC on top of the fibrin matrix were detached and floated in the medium.
At lower concentrations, all the tested compounds showed an inhibitory effect on the VEGF/TNFα-induced tube formation, but the effective concentration window of the compounds was very narrow.
9. Conclusions
Compound B6 seems to be the most potent compound with regard to the inhibition of tube formation. However, compound A4 is also very interesting since this compound didn’t have an effect on HUVEC proliferation but could inhibit tube formation. This may indicate that the working mechanism of this compound may differ when compared to the other compounds.
All these data indicate the simmondsin derivatives are able to inhibit endothelial cells proliferation and tube formation, but more in vitro and in vivo experiments in different assays have to be performed to find out the exact working mechanism of the inhibitory capacity.
[/vc_column_text][/vc_column_inner][/vc_row_inner][vc_row_inner][vc_column_inner width=”1/2″][vc_single_image image=”1562″ img_size=”full”][vc_column_text]Figure 3. Inhibition of VEGF-A-induced HUVEC proliferation by D’Oosterlynck R&D compounds.
Non-confluent HUVEC were cultured for 48 h in the absence or presence of bFGF, VEGF-A, or VEGF-A in combination with the indicated simmondsin derivatives (in w/v) in M199 supplemented with 10% NBCS. After 48 h, a tracer amount of [3H]thymidine was added to the medium and the incubation continued in the same medium for another 6 h and [3H]thymidine incorporation was determined as described in “Characterization of the test systems”. The data are expressed as mean ± SEM of triplicate wells.
[/vc_column_text][/vc_column_inner][vc_column_inner width=”1/2″][vc_single_image image=”1563″ img_size=”full”][vc_column_text]Figure 4. The effects of simmondsin derivatives on in vitro tube formation in 3-D fibrin matrices by hMVEC.
HMVEC were cultured on top of a 3-D fibrin matrix in M199, 10% human serum, 10% NBCS supplemented with VEGF-A (25 ng/mL) and TNF(10 ng/mL) in the presence of a plasmin inhibitor trasylol, 100 U/ml), or the indicated concentrations (w/v) of simmondsin derivatives. After 7 days of culture total tube length (mm/cm2) was measured using image analysis equipment as described in Material and Methods. The data are expressed as the mean with the SEM of VEGF/TNFα-induced tube formation (calculated as described in the Material and Methods) of two wells indicated by the error bars. * = computer measurement is not correct. There were no tube-like structures present, but the hMVEC on top of the fibrin layer were rounded up and measured as being tubes-like structures.
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