| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 28, 2001 as doi:10.1096/fj.01-0427fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Departamento de Biología Molecular y Bioquímica and
* Departamento de Biología Animal, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, 29071-Málaga, Spain
2Correspondence: Departamento de Biología Molecular y Bioquímica, Facultad de Ciencias, Universidad de Málaga, Campus de Teatinos, 29071-Málaga, Spain. E-mail: quesada{at}uma.es
SPECIFIC AIM
In the course of a screening program to identify new antiangiogenic compounds from marine organisms, aeroplysinin-1 was selected. The inhibition of angiogenesis in vitro by aeroplysinin-1 has been characterized in a variety of experimental systems representing the sequential events of the angiogenic process. The ability of aeroplysinin-1 to inhibit angiogenesis in vivo was also investigated in the chick chorioallantoic membrane and the Matrigel plug assays.
PRINCIPAL FINDINGS
1. Aeroplysinin-1 inhibits the capillary tube formation by endothelial cells
The final event during angiogenesis is the organization of endothelial cells in a 3-dimensional network of tubes. In vitro, endothelial cells plated on Matrigel align themselves forming cords, already evident a few hours after plating (Fig. 1
). The addition of aeroplysinin-1 (0.7 µM) to the assay resulted in complete inhibition of BAE cell alignment and cord formation. When HUVE cells were used in the tube formation assay, 3 µM aeroplysinin-1 completely inhibited their in vitro differentiation on Matrigel (Fig. 1)
.
|
2. Aeroplysinin-1 inhibits the growth of endothelial cells
Angiogenesis involves local proliferation of endothelial cells. We investigated the ability of aeroplysinin-1 to inhibit the proliferation of actively growing endothelial cells. Aeroplysinin-1 inhibited, in a concentration-dependent manner, the growth of cultured BAE cells. IC50 value of this anti-proliferative effect was 2.1 ± 0.2 µM (mean of three different experiments±SD), for subconfluent BAE cells stimulated to grow with 10% FBS. The activity of this compound decreased when low proliferant endothelial cells were used. In high density-low proliferant BAE cells, the IC50 value for aeroplysinin-1 significantly increased to 7.30 ± 3.2 µM (mean of three different experiments±SD P<0.05).
3. Aeroplysinin-1 induces apoptosis in activated endothelial cells
Our results show that apoptosis was induced in BAE cells by aeroplysinin-1 (3 µM) treatment for 14 h as determined by TUNEL staining.
4. Aeroplysinin-1 induces a shift in the proteolytic balance toward anti-proteolysis
Gelatin zymography of conditioned media and cell extracts of BAE cells untreated and treated for 24 h with 3 µM of aeroplysinin-1 shows that the concentration of MMP-2 in the medium conditioned by aeroplysinin-1-treated cells is clearly lower than that of untreated cell medium. We have also observed that the treatment with 3 µM aeroplysinin-1 affects the balance of PA/PAI levels, another system involved in extracellular matrix remodeling in angiogenesis. Aeroplysinin-1 induced an important decrease in the expression of PA protein and a parallel increase in the expression of PAI protein in both conditioned media and cell extracts. Altogether, the results clearly show that aeroplysinin-1 induces a shift in the proteolytic balance toward anti-proteolysis.
5. Aeroplysinin-1 inhibits migration and invasion of BAE cells
The effects of aeroplysinin-1 on fluorescence-labeled BAE cell migration and invasion through Matrigel-coated filters were investigated. As shown in Fig. 2
, aeroplysinin-1 inhibits both processes in a dose-dependent manner. 1.5 µM aeroplysinin-1 completely abolished migration of BAE cells in the modified Boyden chamber assay (Fig. 2A
). The presence of 3 µM aeroplysinin-1 during the chemoinvasion assay resulted in a 50% inhibition of the ability of BAE cells to invade through Matrigel-coated filters (Fig. 2B
).
|
6. Aeroplysinin-1 inhibits angiogenesis and induces apoptosis in the chick chorioallantoic membrane
Treatment with aeroplysinin-1 caused a dose-dependent antiangiogenic effect with 27% of treated eggs scored as positive at 1.5 nmol and had a maximum effect at 3 nmol, where 100% of the eggs scored positive.
Aeroplysinin-1 showed a potent apoptotic activity in the CAM membrane, as shown by the TUNEL assay. Apoptosis was detectable 19 h after treatment; the effect was located primarily in the areas of vascular differentiation (i.e., the chorioallantoic surface) and in the walls of the large vessels. The effect on the endothelium and blood islands filled with immature blood cells was particularly intense, as shown in QH1/TUNEL double labeling of the quail CAMs. Other cells, such as fibroblasts or the endodermal allantoic cells, remained largely unaffected by the treatment.
7. Aeroplysinin-1 inhibits angiogenesis in the Matrigel plug assay
In the Matrigel plug assay, aeroplysinin-1 caused a strong inhibition of the cell invasion. All the parameters assessed by image analysis (i.e., percentage of surface covered, number and scattering of particles) were significantly different between control and aeroplysinin-treated Matrigel plugs, with an average reduction of 
70%. The difference was especially remarkable in the number of particles and in their spreading throughout the plug volume. The density of factor VIII-related antigen-immunoreactive vessels and the number of proliferating cells (assessed by PCNA staining) were greatly reduced in the aeroplysinin-treated Matrigel plugs in relation to controls. The average reduction was 90% as assessed by image analysis. Peripheral areas surrounding the Matrigel plug showed abundant PCNA-positive cell, in control and experimental mice.
CONCLUSIONS
The development of new blood vessels is a complex multistep process. Endothelial cells resting in the parent vessels are activated by an angiogenic signal and stimulated to synthesize and release degradative enzymes allowing endothelial cells to migrate, proliferate, and finally differentiate to give rise to capillary tubules. Any of these steps may be a potential target for pharmacological intervention. The present study demonstrates that aeroplysin-1 inhibits several essential steps of the angiogenic process (Fig. 3
).
|
Antiangiogenic activity of aeroplysinin-1 was first detected using the in vitro differentiation assay for endothelial cells. Our results show that aeroplysinin-1 inhibits capillary-like tube formation by BAE or HUVE cells (Fig. 1)
. We have also shown that aeroplysinin-1 inhibits the growth of highly proliferating BAE cells more efficiently than that of low-proliferant BAE cells, which suggests the possibility that aeroplysinin treatment could affect mainly the newly formed rather than preexisting blood vessels. The results from the TUNEL assay suggest that the cytotoxic effect produced by aeroplysinin-1 on proliferant endothelial cells could be due at least in part to an induction of apoptosis. A positive proteolytic balance is required for capillary sprout elongation and lumen formation during angiogenesis. Our data indicate that aeroplysinin-1 causes a shift toward anti-proteolysis in endothelial cells. This agrees with the observed inhibition of the migration and invasive ability of the aeroplysinin-treated BAE cells (Fig. 2)
.
Inhibition by aeroplysinin-1 of the mentioned essential steps of in vitro angiogenesis agrees well with the effect on in vivo angiogenesis we observed in the CAM model. We have also seen that aeroplysinin-1 produces a devastating effect on developing vessels of the chorioallantoic membrane, probably due to induction of apoptosis in the vascular cells and their progenitors. On the other hand, aeroplysinin provoked an inhibition of the bFGF-mediated cell invasion in the intradermal Matrigel plug model. The inhibition was not complete, and some cells were found within the aeroplysinin-treated Matrigel. However, unlike the controls, the invading cells rarely showed factor VIII-related antigen, a differentiation marker of endothelial cells. A possible explanation could be that aeroplysinin-1 specifically blocked the migration and/or differentiation of endothelial cells within the Matrigel, as observed in the in vitro assays. Finally, the proliferation marker PCNA was rarely found in cells within the experimental Matrigel, in contrast to the large number of PCNA+ cells observed in control plugs, indicating that aeroplysinin-1 either inhibited proliferation of the cells that invaded the Matrigel or precluded proliferating cells to invade the plug.
In summary, data shown here indicate for the first time that aeroplysinin-1 is a compound that interferes with key events in angiogenesis, making it a promising drug for further evaluation in the treatment of angiogenesis-related pathologies.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0427fje; to cite this article, use FASEB J. (December 28, 2001) 10.1096/fj.01-0427fje 
This article has been cited by other articles:
|
|
 |
|
  B. Martinez-Poveda, R. Munoz-Chapuli, S. Rodriguez-Nieto, J. M. Quintela, A. Fernandez, M.-A. Medina, and A. R. Quesada IB05204, a dichloropyridodithienotriazine, inhibits angiogenesis in vitro and in vivo Mol. Cancer Ther., October 1, 2007; 6(10): 2675 - 2685. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. E. G. Muller, N. L. Thakur, H. Ushijima, A. N. Thakur, A. Krasko, G. Le Pennec, M. M. Indap, S. Perovic-Ottstadt, H. C. Schroder, G. Lang, et al. Matrix-mediated canal formation in primmorphs from the sponge Suberites domuncula involves the expression of a CD36 receptor-ligand system J. Cell Sci., May 15, 2004; 117(12): 2579 - 2590. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or 1.5 µM (
) aeroplysinin-1. DMEM/10% FBS was used as chemoattractant in the lower wells. The inserts were incubated at 37°C and cell migration was determined by readings at the times indicated. B) Invasion assay. 2 x 105 fluorescence-labeled BAE cells suspended in DMEM/0.1% BSA were incubated (4 h, 37°C) on top of Matrigel-coated filters in the absence or presence of aeroplysinin-1. DMEM/10% FBS was used as chemoattractant in the lower wells. The results are the means ± SD of triplicate filters. a) Statistically significant difference (P<0.05); b) statistically significant difference (P<0.01); c) statistically significant difference (P<0.005) between aeroplysinin-treated cells and their respective untreated control cells.