Antiangiogenic effect of betamethasone on the chick cam
stimulated by TA₃ tumor supernatant

DAVID LEMUS¹, ALFREDO DABANCENS², JULIO ILLANES¹, MARCELA FUENZALIDA¹, ANIBAL GUERRERO² and CLAUDIA LÓPEZ³

Programa de Morfología¹, Instituto de Ciencias Biomédicas (ICBM) Facultad de Medicina, Universidad de Chile, Santiago, Chile
Instituto de Anatomía Patológica², Hospital Clínico, Universidad de Chile
Instituto de Salud Pública de Chile³

Correspondence to: David Lemus. Programa de Morfología, ICBM Facultad de Medicina. Independencia 1027, Correo 7, Casilla 70079 Santiago 7. Fax: (562) 678-6264, . e-mail: dlemus@machi.med.uchile.cl

Received: May 14, 2001. Accepted: September 11, 2001

ABSTRACT

Tumor growth is the result of combined cell proliferation overwhelming cell death and neoangiogenesis. This report shows CAM angiogenesis promoted by TA₃ tumor supernatant with or without low dosis of betamethasone (Minimal antiangiogenic concentration: ß-MAAC).
Methylcellulose discs instilled with 10 µl of ß-MAAC (0.08 µg/ml),10 µl of tumor supernatant(TA₃ts),5 µl ß-MAAC + 5 µl TA₃ts, and 10 µl of PBS as control were implanted in host chick eggs. On day 12, the grafts were removed, photographed and fixed. Sections were stained in parallel, one and three with hematoxylin-eosin, and section two by the Tunel method. The number of vessels was evaluated in a microscopic field of the CAM (2250 µm² ). The results show that ß-MAAC produced a significant inhibition of neovascularization in comparison to that observed in controls (P < 0.0025; Student t-Test). Discs instilled with TA₃ts produced an intense stimulation of angiogenesis in contrast, when discs were instilled with 5 µl of ß-MAAC + 5 µl of TA₃ts the angiogenesis was significantly inhibited (P< 0.001). The results show that effective antiangiogenic doses of betamethasone are in the range of 10^-7 M, (probably a genomic mediated action) and that this effect of low concentration may have clinical applications. (Biol Res 2001; 34 3-4: 227-236)

Key words: antiangiogenesis, betamethasone, CAM, tumor

INTRODUCTION

Angiogenesis is a complex multi-step process comprising a series of cellular events that lead to neovascularization from existing blood vessels. It is associated with the processes of inflammation, wound healing, tumor growth, and metastasis (Folkman, 1995; Pluda, 1997; Nangia-Makker et al., 2000). Determination of microvessel density in a growing cancer has been shown to have prognostic value for recurrence and survival (Vartanian and Weider, 1994).

In recent years relevant studies have focused on identifying angiogenesis stimulators, leading to the identification of several angiogenic factors. These can be divided into three groups of extracellular signals. The first one comprises soluble growth molecules such as acid and basic fibroblast growth factors (aFGF and bFGF respectively) and vascular endothelial growth factor (VEGF), which affect endothelial cell growth and differentiation (Burgess and Macaig, 1989; Conn et al., 1990). The second group of factors inhibits proliferation and enhances differentiation of endothelial cells and includes transforming growth factor ß(TGFß), (Muller et al., 1987; Pepper et al., 1999), angiogenin, as well as several low-molecular-weight substances (Folkman and Ingber., 1987; Meininger et al., 1992). The third group comprises extracellular matrix-bound cytokines released by proteolysis, which may contribute to angiogenic regulation (Meininger et al., 1992).

It has been demonstrated that tumors can also generate inhibitors of angiogenesis, including angiostatin (O'Reilly et al., 1994), trombospondin (Dipietro and Polverini, 1993), endostatin (O'Reilly et al., 1997). Additionally, a number of tumor-associated macrophages that secrete ßFGF, tumor necrosis factor a (TNF-a), endothelial growth factor (EGF), and VEGF, and other cytokines, have been shown to play a role in tumor angiogenesis (Polverini, 1989). This evidence points to the notion that angiogenesis is governed by a balance between positive and negative regulators within the microenvironment (Folkman, 1992; Iruela-Arispe and Dvorak, 1997).

The chorioallantoic membrane (CAM) has been utilized as an in vivo system to study angiogenesis, antiangiogenesis, and teratogenic effects on the chick embryo. Many investigators have studied the histological and morphological changes associated with the proliferation of new vessels and tumor neovascularization by direct observation using CAM (Folkman and Ingber, 1987; Mostafa et al., 1980; Petruzzeli et al., 1993; Quigley and Armstrong, 1998; Ribatti et al., 1996a; Gavrieli et al., 1992; Folkman et al., 1983; Buttgereit et al., 1998; Illanes et al., 1999). For example, Folkman and Ingber, (1987) reported a new class of angiostatic steroids that circulate in the plasma. This new steroid function was revealed from a series of experiments on the role of heparin in angiogenesis. In fact, application of cortisone or hydrocortisone to the CAM prevented the inflammatory reaction but did not interfere with tumor-induced angiogenesis.

In a histological study of CAM blood vessels with methylcellulose discs instilled on the tenth day of incubation with TA₃ tumor supernatant (TA₃ts), we observed a rapid capillary proliferation at the site of the implant on the twelfth day of incubation. Based on these findings, our current work analyzes the relationship between CAM angiogenesis promoted by TA₃ tumor supernatant and betamethasone in minimum antiangiogenic active concentration (ßmet-MAAC).

MATERIALS AND METHODS

In ovo procedure

The host CAM was prepared as follows: Fertilized White Leghorn chick eggs supplied by the Institute of Public Health were employed for this study. The eggs were incubated in humidified atmosphere at 38.5ºC. At 48 hours of incubation, a square window was opened into the shell after removing 2 ml of albumen so that the developing CAM became detached from the shell and the underlying CAM vessels were disclosed. The opening was closed with cellotape, and the incubation carried on until the day of the experiment. The primitive CAM vessels continued to proliferate and differentiate into an arteriovenous system until day 8, thus giving rise to a network of capillaries that migrated to occupy an area beneath the chorion. Rapid capillary proliferation occurred until day 12, after which the mitotic index of the CAM vessels declined rapidly, and the vascular system attained its final arrangement on day 18, just before hatching.

The TA₃ tumor, provided by the Pathology Program, ICBM Institute, is a local tumor growth of a cell line transplanted into mice. The mice were anesthetized with ether and sacrificed, and their tumors were then surgically isolated and centrifuged at 1.085 g for 15 min. The pellet obtained was discarded, and the tumor supernatant (TA₃ts) was utilized for further analysis.

 

Evaluation of the angiogenic response of CAM to: discs instilled with angiogenic and antiangiogenic agents

On day 8 of development, previously sterilized methylcellulose discs (5 mm diameter, 0.25 µm pore size, 125 µm thick) were implanted on the surface of the growing CAM to study the vessels' inflammatory angiogenesis response. In previous research we established the minimum antiangiogenic active concentration of betamethasone (ßmet-MAAC) (phosphate sodium salt minimum 97%, FW 516.4, Sigma Chemical Company, P.O. Saint Louis MO, USA), by instilling different concentrations of betamethasone on the discs in CAM (unpublished data). Those experiments showed that 10 µl [0.08 µg/ml] of betamethasone produced the minimum inhibition of the angiogenesis in CAM. The instillation of eggs was performed as previously reported by Illanes et al., (1999). On the tenth day of incubation, control discs were instilled with 10 µl of PBS. Some of the remaining eggs were instilled with 10 µl of ßmet-MAAC, others with 10 µl of TA₃ts, and the rest with a combination of 5 µl of ßmet-MAAC and 5 µl of TA₃ts (Fig. 1). Host eggs were further incubated for 48 hours. At the end of the culture period (12 days) the grafts were removed, photographed and fixed in 10% formaldehyde. For light microscopy, the material was immersed in increasing concentrations of ethanol and infiltrated with paraffin for four periods of 20 min each with changes of paraffin (m.p. 58-60ºC). The tissue was sectioned at 5 µm-thickness. Five independent observers analyzed the CAM's reaction to the discs, which consisted of either forming new vessels or inhibiting their formation in response to the soluble factors utilized. The number of vessels was evaluated as the total number in a microscopic field of the CAM 2.250 µm² utilizing a square reticule. The evaluation of the number and density of blood vessels was processed statistically.

 

Figure 1: Schematic representation of the experimental procedure. Fertilized hen eggs were incubated for 12 days in a humidified incubator at 37.8°C. A.) After 48 hours of incubation a smaller hole was ground down over the air sac (short arrow). Arrowhead points to extraction 2 ml of albumen, and long arrow shows the square window realized (n=120). B.) After 8 days of incubation plain filter discs with were implanted on the CAMs (n=110). After 10 days of incubation: C1) Instilled discs with PBS (control disc). C2) Instilled discs with 10 µl of betamethasone in 1.5 x 10-7 moles/liter [0.08 µg/ml] in minimum antiangiogenic active concentration (bmet-MAAC). C3) Instilled discs with 10 µl of TA3 tumor supernatant (TA3ts). C4) Instilled disc with 5 µl ßmet-MAAC + 5 µl TA3ts. After 12 days of incubation the quantification of vascular density of CAM was calculated in 10 lineal mm and in detection of apoptotic cells (n=59).

 

Three sections of CAM were stained in parallel in each experiment. In order to find the main angiogenesis zone, sections one and three in the samples studied were stained with hematoxylin-eosin. Section two was tested by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method (Gavrieli et al., 1992) for identification of programmed cell death (PCD). Nuclei of tissue sections were stripped from proteins by incubation with 20µg/ml proteinase K (PK)(Sigma Chemical Co.) for 15 min at room temperature (RT), and the slides were then washed four times in double-distilled water (DDW) for 2 min each. Endogenous peroxidase was inactivated by covering the sections with 2% H₂O₂ for 5 min at RT. The sections were rinsed with DDW and immersed in TDT buffer (30 mM Trizma base, pH 7.2, 140 mM sodium cacodylate, 1 mM cobalt chloride). TDT (0.3 e.u./µl) and biotinylated dUTP in TDT buffer were then added to cover the sections and then incubated in a moist environment at 37ºC for 60 min. The reaction was terminated by transferring the slides to TB buffer (300 mM sodium chloride, 30 mM sodium citrate) for 15 min at RT. The sections were rinsed with DDW, covered with 2% aqueous solution of bovine serum albumin (BSA) for 10 min at RT, rinsed in DDW, and immersed in PBS for 5 min. The histological sections were covered with extra-avidin peroxidase (Promega Corporation Madison, Wi, USA, 608-274-4330) diluted 1:10-1:20 in DDW, incubated for 30 min at 37ºC, washed in DDW immersed for 5 min in PBS, and stained with ABC complex for 30 min at 37ºC. The evaluation of the number and density of vessels was evaluated by five independent observers. The total number of eggs employed in this study was 120, of which 25 were used in each treatment (Table I).

 

Table I
Number of eggs employed in each treatment

Day	PBS	ßmet-MAAC	TA 3ts	ßmet-MAAC+TA 3ts	Total
2 8		Host Cam Implanted Discs			120 110
10	25	25	25	25	100
12	15	11	18	15	59

RESULTS

Angiogenesis appears to depend on the balance of several stimulating and inhibiting factors. The inhibition of blood vessel growth, i.e. antiangiogenesis, or the stimulation of angiogenesis-inhibiting factors seem to provide a strategy for preventing both the growth of tumors and other angiogenesis-dependent diseases.

Our results show that methylcellulose discs instilled with 10 µl [0.08 µg/ml] of betamethasone in the CAM showed a significant inhibition of embryonic neovascularization in the zone of the filter. This effect was observed in all the eggs tested, while control CAMs developed major vascular zones (Figures 2A, 3A, 4A, and 2B, 3B, 4B). The evaluation of the number of capillaries in histological sections of the CAM at 12 days of incubation showed a mean of 7 ± 0.40 vessels in 2.250 µm². Under the same conditions, the number of capillaries increased to 8.78 ± 0.38 when the discs plus 10 µl PBS were instilled in the CAM; this effect represents an inflammatory response to a foreign body. Discs instilled with 10 µl of betamethasone (ßmet-MAAC) produced a marked decrease in capillaries in 2.250 µm² equal to 5.35 ± 0.30 (p < 0.002), Student t-test. On the other hand, discs instilled with 10 µl of supernatant (TA₃ts) produced an intense stimulation of the angiogenesis in all the eggs tested: 10.15 ± 0.61 capillaries in the same determined microscopic field of the CAM (2.250 µm²). In contrast, when discs were instilled with a combination of 5 µl of betamethasone (ßmet-MAAC) and 5 µl of supernatant (TA₃ts, the number of capillaries decreased to 8.24 ± 0.39, (p< 0.001), Student t-test. (Fig. 5).

 

DISCUSSION

It has been described elsewhere that cortisone transforms the angiogenic promotion properties of heparin in an antiangiogenic effect, and furthermore, that cortisone alone has been shown to have little or no effect (Folkman et al.,1983).

 

Figure 5 : Summary of angiogenic and antiangiogenic response of CAM at 12 days of incubation to discs instilled with: 10 µl of PBS (PBS-disc, control); 10 µl of minimum antiangiogenic active concentration of betamethasone (ßmet-MAAC, [0.08 µg/ml]); 10 µl TA3ts of supernatant (TA3ts), and a combination of 5 µl of ßmet-MAAC and 5µl of supernatant of tumour TA3. Bars represents the mean ± SEM of 50 of each.

 

The antiangiogenic effect of that combination was independent of the type of angiogenic stimulus. This is also true for tumor angiogenesis on chick embryo CAMs, for embrionic angiogenesis in chick embryo yolk sacs and CAMs, and for inflammatory and immune angiogenesis. Hexasaccharide heparin fragments without anticoagulant activity obtained by heparinase were angiostatics in combination with 100 µg cortisone.

The same combination produces an anti-tumoral effect using daily subcutaneous doses of 73 mg/kg of cortisone with 200 - 1,000 U/ml of heparin. Several tumors (100% of reticulum cell sarcomas, 100% of Lewis lung carcinomas, and 80% of B-16 melanomas) experienced "complete regression" between these doses, although tumor growth continued higher concentrations of heparin. Other tumors, such as gliomas and Sarcoma 1509 did not regress. When hydrocortisone, methylprednisolone, dexamethasone and medroxyprogesterone in combination with heparin were compared, only hydrocortisone proved as effective as cortisone. Dexamethasone at 3.2 mg/kg was ineffective with or without heparin.

In 1987 Folkman and Ingber reviewed the subject of angiostatic steroids, mentioning that they could be glucocorticoids or mineralocorticoids joined to heparin or fragments of heparin. Cortisone, hydrocortisone, 11ª-hydrocortisone and tetrahydrocortisol were the most active. The dissolution of the basal membrane of the newly formed capillaries was the proposed mechanism of action.

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. There are also GRßs that are unable to bind to hormones, and which function as the antagonist of the a form. Other genes are thought to be inhibited by the glucocorticoid GR association, probably through indirect protein-to-protein interactions between the GR and the transcriptional factors of these genes (Boumpas, 1996; Haddad et al., 1996; Yang-Yen et al., 1990).

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). These last two mechanisms explain some of the nearly immediate pharmacological effects that occur within seconds after high doses of glucocorticoids (Inagaki et al., 1992), but the physiological effect at very low concentrations is mediated by genomic mechanisms and requires at least 20 - 30 minutes to take place.

Many of the anti-inflammatory effects of glucocorticoids are mediated through the production of lipocortine 1 (Goulding and Guyre, 1993), which inhibits the A2 Phospholipase (Barnes and Karin, 1997). Other mechanisms are mediated by negative GR interaction with the transcriptional factor of the Phospholipase A2 gene. There is no definitive explanation of the glucocorticoid antiangiogenic mechanism.

The antiangiogenic effect of Betamethasone demonstrated here has not been previously reported. The participation of heparin or heparin fragments is unlike, because no mast cells appeared in CAMs during vasculogenesis. The antiangiogenic effect of betamethasone was confirmed in rats through the subcutaneous polyurethane implantation model (results not shown). Because the dosage needed to obtain an antiangiogenic effect is in the range of 10^-9 M, it is most probable that this is a genomic mediated action.

Research continues to analyze the differences between dexamethasone and betamethasone, two molecules with the same chemical composition and differing only in the cis or trans position of the 16 methyl group. The results reported here on the antiangiogenic effect of low betamethasone concentration may have clinical applications.

ACKNOWLEDGEMENTS

This investigation was supported by the Chilean National Science Foundation: Fondo Nacional de Investigación Científica y Tecnológica Grant 1990852.

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