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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.35 n.3-4 Santiago  2002

http://dx.doi.org/10.4067/S0716-97602002000300008 

Biol Res 35: 339-345, 2002

 

Effects of betamethasone, sulindac and quinacrine drugs
on the inflammatory neoangiogenesis response induced
by polyurethane sponge implanted in mouse

JULIO ILLANES1, ALFREDO DABANCENS2, OLGA ACUÑA3, MARCELA
FUENZALIDA
1, ANIBAL GUERRERO1, CLAUDIA LOPEZ4 AND DAVID LEMUS1.

1-Programa de Morfología, Instituto de Ciencias Biomédicas (ICBM) Facultad de Medicina,
Universidad de Chile, Santiago-Chile.
2-Instituto de Anatomía Patológica, Hospital Clínico, Universidad de Chile.
3-Departamento Biomédico, Facultad Ciencias de la Salud, Universidad de Antofagasta.
4-Instituto de Salud Pública de Chile.

ABSTRACT

In this study, we showed the effect of the betamethasone, sulindac and quinacrine alone or combined, on the inflammatory angiogenesis promoted by polyurethane sponge on mice. The main finding reported here is that the formation of new blood vessels was strongly inhibited by low concentration of betamethasone, sulindac or quinacrine, whether alone or in combination. It is known that steroidal anti-inflammatory drugs inhibit the enzymes required for the production of prostaglandins through a nuclear glucocorticoid receptor (GR) mediated mechanism. This mechanism may occur in endothelial cells as well. Considering that activity of cyclo-oxigenases 1 and 2 is inhibited by sulindac, and that these enzymes are located in the stromal tissue, we propose that the anti-angiogenic effect of these agents may occur via inhibition of both COX isoforms. On the other hand, quinacrine inhibited PLA2 activity, and we propose here that the anti-angiogenic effect occurs via inhibition of the enzyme PLA2. The potentiated effect of the association of betamethasone, sulindac and quinacrine may have some therapeutic benefit in the control of pathological angiogenesis. Further studies are required to validate these propositions.

Key terms: angiogenesis, antiangiogenesis, betamethasone, sulindac, quinacrine

INTRODUCTION

Angiogenesis is the development of new blood vessels and capillaries from the existing vascular bed. Under normal conditions, this tightly regulated process occurs only during embryonic development, the female reproductive cycle, and wound repair. This process is regulated by a balance between pro- and anti-angiogenic molecules and is derailed in various diseases, especially cancer. It is now widely accepted that when the effect of pro-angiogenic molecules is balanced by that of anti-angiogenic molecules, the angiogenesis is stopped, and when the net balance is tipped in favor of angiogenesis, the process is activated (Malonne et al.,1999; Hanahan and Weinberg, 2000; Bouck et al., 1996). On the other hand, normal angiogenesis and development are controlled by the balance between cell proliferation and apoptosis, and there is strong evidence that tumor growth is not just a result of uncontrolled proliferation but also of reduced apoptosis (Reed, 1999; Tamm et al., 2000). Several anti-angiogenic agents alone or in combination with conventional therapies are now in clinical trials. These trials are based on strategies that a) interfere with angiogenic ligands, their receptors or downstream signaling; b) upregulate or deliver endogenous inhibitors, or c) directly target tumor vasculature (Carmeliet, 2000). These approaches offer new hope for the successful treatment of cancer. However, there are a number of potential problems that warrant caution in clinical trials on humans.

Our present work describes the effect of the anti-inflammatory drugs beta-methasone, sulindac and quinacrine, administered either alone or associated on mice inflammatory angiogenesis promoted by a polyurethane sponge matrix implanted subcutaneously in the dorso-cervical region.

MATERIALS AND METHODS

Mice: 45 female AJ strain mice were used at 8 weeks of age and weighing 25 g each. The mice were maintained in caged groups of 5 animals at 25 to 26 ºC and fed pellets and ad-libitum water. The animals were premedicated intraperitonally with diazepam (1.0 mg/kg). After 5 min, the induction phase with ketamine (100 mg/kg) and atropin (0.1 mg/kg) was carried out for the same way. Polyurethane sponges were cut into 5 x 5 x 5 mm pieces and prepared according to Boyle and Mangan (1982). Briefly, sponge pieces were rinsed in distilled water and sterilized in an autoclave at 2 atmosphere for 30 min. The PBS-impregnated sponges were implanted subcutaneously (s.c.) surgically via a dorso-cervical incision. The skin wound was sutured with surgical nylon. All procedures were performed under anesthesia and sterile conditions.

The experimental design is outlined in Figure 1. In this standard model, one sponge piece was implanted in the dorso-cervical site. Five days after implantation, the mice were inoculated intraperitonally with betamethasone (10 µg/animal), sulindac (5 µg/animal) and quinacrine (12.5 mg/animal), either alone or combined in a volume of 100 µl per animal. A summary of the drug concentrations used in the different treatments is presented in Table I. All experimental procedures were carried out following the animal handling regulations established by the University of Chile Medical School.

Figure 1. Schematic outline of the standard protocol for in situ activation of inflammatory neoangiogenesis response induced by polyurethane sponge in mice.

On the sixth day, the mice were anesthetized with ether and killed. The sponges were surgically removed and fixed in 10% formaldehyde. The material was processed in an autotechnicon apparatus through increasing concentrations of ethanol and infiltrated in paraffin (m.p. 58-60 ºC). A common protocol was followed for all experiments. In order to establish the primary zone of angiogenesis in the sponge, 5-µm histological sections were obtained, four of which were processed in parallel. Sections 1 and 4 were stained with hematoxylin-eosin, section 2 was processed using apoptotic cell labeling (Gavrieli et al., 1992), and anti-CD31 (endothelial cell labeling) was applied to section 3.

Three independent observers analyzed the reaction of the mice to the implant. The number of blood vessels was evaluated as the total number of capillaries in an area equivalent to 22,500 µm2 from an eye-piece reticule lens at 400X. A total of 40 mice were employed in this experiment, 5 in each treatment. Table II shows the total number of histological sections examined per treatment. Comparisons of MCD between the control and the different conditions were performed using ANOVA Test and the Bonferroni Multiple Comparisons Test.

RESULTS

To determine whether the intraperitonal administration of steroidal and nonsteroidal drugs inhibited inflammatory angiogenesis, we examined the vessel density in histological sections from treated and control mice subsequent to immunostaining with antibodies against CD31 (Figures 2, 3). Significant differences in mean vessel counts were found between control and treated mice. In fact, six days after the implantation of polyurethane sponge with PBS, the treated mice showed an MCD of 3.15 ± 0.18 vessels, which represents an inflammatory response to foreign body.

Figures 2 and 3 show histological cross sections of sponge surgically removed from mice six days after implantation. Thick arrows point to immunohistochemical staining for CD31 expressed on capillary endothelial cells. Thin arrow points to sponge matrix 100 X.

Figure 3. Higher magnification of endothelial cell labeling anti-CD31. Intense positivity is observable in endothelial cells (thick arrows) (400X).

Regarding the effects of steroidal (betamethasone), NSAI drugs (sulindac) and (quinacrine) inhibitor of phospholipase A2 drug , we observed that mice treated with these agents displayed a drastic decrease in the mean capillary density (MCD) (0.00±0.00 per area for betamethasone; 0.02±0.01 for sulindac and 0.00±0.00 for quinacrine) compared with the PBS inflammatory angiogenesis control. Similar effects were observed when the association of three agents was analyzed: 0.05±0.01 for betamethasone+sulindac, 0.02±0.01 for betamethasone+quinacrine, 0.02±0.01 for sulindac+quinacrine, and 0.01±0.01 for betamethasone + sulindac + quinacrine. It must be pointed out that the anti-angiogenic effect of betamethasone and quinacrine was more pronounced. This is depicted in Figure 5. All comparisons were highly significant (p<0.001, see Table II).

Figure 5. Summary of efficacy differences among several angiogenesis inhibitors. Seven angiogenesis inhibition treatments on mice at six days of sponge matrix implantation are compared here: (PBS) control; (PBS+ß) PBS+betamethasone; (PBS+S) PBS+sulindac; (PBS+Q) PBS+quinacrine; PBS+ß+S; PBS+ß+Q; PBS+S+Q; PBS+ß+S+Q. Bars represent the mean ± SEM.

Light microscopy observation of histological sections from mice implanted with the sponge matrix with sulindac revealed the presence of same endothelial and stromal cells showing apoptosis (Figure 4).

Figure 4. Apoptotic endothelial and mesenchymal cells staining for TUNEL (thick arrows) in paraffin-embedded tissue sections counterstained with Hematoxylin-eosin (400X).

DISCUSSION

The primary finding in the present work is that the formation of new blood vessels, induced by polyurethane sponge impregnated with PBS, was highly inhibited in the presence of low concentrations of betamethasone, sulindac and quinacrine, whether alone or combined. The anti-angiogenic effect of these drugs on new blood vessels that originated as a result of the inflammatory process induced by the polyurethane sponge could be significant. In fact, new, proliferating capillaries have fragmented basal membranes and are leaky, making them more penetrable by tumor cells than mature vessels are (Nagy et al., 1989). Furthermore, the invasive chemotactic behavior of growing capillaries is facilitated by their secretion of endothelial cells at the tips of collagenases and plasminogen activator (Moscatelli et al., 1981). However, the mechanisms through which these drugs could regulate the growth of endothelial cells on sponges implanted in mice are still not fully understood.

According to Buttgereit et al. (1998), glucocorticoids exert their actions through three different mechanisms: a) genomic; b) non-genomic receptor-mediated actions; and c) non-genomic physicochemical actions. The genomic mechanisms are believed to be direct; the glucocorticoids binds to cytosolic glucocorticoid receptors (GRa) that interact with specific "glucocorticoid-responsive elements" in the DNA (Bamberger et al., 1995), promoting the transcription of genes. Lemus et al (2001) reported a direct anti-angiogenic effect of betamethasone in the range of 10-9 M, which is probably a genomic mechanism on the chick CAM.

The non-genomic mechanism can be triggered through membrane receptors (Moore and Orchinik, 1994) or through physicochemical modifications of the membrane (Pietras and Szego, 1975). Of greatest importance for the anti-inflammatory action of glucocorticoids is lipocortin 1, which inhibits phospholipase A2 on the arachidonic acid cascade (Goulding and Guyre, 1993) by cyclooxygenases and lipooxygenases, which in turn inhibits the synthesis of mediators of inflammation, such as prostaglandins. Prostaglandins are formed via the cyclooxygenase (COX) pathway and are known to be potent proangiogenic molecules (Needleman et al., 1986; Levy, 1997). We observed the anti-angiogenic effect of betamethasone alone or combined with sulindac or quinacrine or both on the doses employed.

Sulindac is a type of NSAID, and is therefore able to inhibit COX1 and COX2 (Williams et al., 2000). In fact, sulindac inhibited cyclooxigenases 1 and 2 (Soh et al., 2001). We therefore speculate that the anti-angiogenic effect of sulindac may occur via the inhibition of COX1 and COX 2 and induce apoptosis. Considering that these COX isoforms may be located in the stromal compartment, sulindac may lead to an inhibition in the production of proangiogenic factors, as is observed in the inflammatory angiogenesis analyzed in this study. On the other hand, sulindac is metabolized in vivo to sulfide and sulfone derivatives. Both the sulfide and sulfone metabolites of sulindac, as well as the more potent cyclic GMP-dependent phosphodiesterase inhibitors, were shown to inhibit extracellular signal-regulated kinase (ERK)1/2 phosphorylation at doses (40-60 microM) and times (1-5 days) consistent with the induction of apoptosis by the drugs (Rice et al., 2001). Sulindac sulfone induces apoptosis and exhibits cancer chemopreventive activity, but in contrast to sulindac, it does not inhibit cycloxygenases 1 or 2 (Soh et al., 2000).

Quinacrine is an inhibitor of phospholipase A2 (PLA2s) (Lapetina et al., 1981). The involvement of PLA2s in inflammation is the result of their ability to mobilize arachidonic acid from phospholipids. Arachidonic acid serves as a substrate for prostaglandin H synthase 1 and 2 (COX-1 and COX-2, respectively), resulting in the production of prostaglandins. Prostaglandins activate cellular receptors, resulting in the subsequent initiation of signal cascades involving G-proteins and cyclic AMP (Cirino, 1998). It has been shown that the expression of VEGF by the host fibroblast plays an important role in angiogenesis (Fukumura et al., 1998), that prostaglandins can regulate VEGF and bFGF expression (Hoper et al., 1997, Cheng et al., 1997), and that COX inhibitors can directly affect angiogenesis (Daniel et al., 1999, Yamada et al., 1999). We postulate that quinacrine may be expected to have a similar angiostatic glucocorticoid effect of inhibiting PLA2, and that this effect may occur by the synergistic action of both drugs. Moreover, there are different PLA2s isoforms that are important in the signaling of several cellular processes and known to play a significant role in inflammation (Glaser, 1995).

In the present study, the effect of betamethasone, sulindac and quinacrine alone or combined effectively decreased inflammatory angiogenesis. These three drugs may acting synergistically by means of anti-angiogenic factor on mice. The study of angiogenic inhibitors is a critical area of research due to the potential pharmacological benefit of these compounds in the treatment of inflammation and cell injury and as a tool for controlling pathological angiogenesis, such as ischaemic and inflammatory diseases and cancer. Further studies are necessary to validate these propositions.

ACKNOWLEDGEMENTS

This research was supported by the Chilean National Science Foundation: Fondo Nacional de Investigación Científica y Tecnológica, Grant No. 1999852.

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Corresponding author: Dr. David Lemus, Programa Morfología, ICBM, Facultad de Medicina. Independencia 1027, Correo7, Casilla 70079. Fax: (56-2) 6786264. e-mail: dlemus@machi.med.uchile.cl

Received: January 30, 2002. In revised form: September 9, 2002. Accepted: September 10, 2002

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