J. Steroid Biochem. Molec. Biol. Vol.42. No. 7, pp. 687-693. 1992
0960-0760/92$5.00+ 0.00 Copyright© 1992PergamonPressLtd
Printedin GreatBritain.All rightsreserved
ANGIOSTATIC CORTISOL
ACTIVITY
IN THE (CAM)
AND
METABOLISM
CHORIOALLANTOIC
OF
THE
CHICK
OF
MEMBRANE
EMBRYO
LORETTAG. McNA'I-r,* DEBORAHLANEand ABBOTF. CLARK Alcon Laboratories, Inc., 6201 South Freeway, Fort Worth, TX 76134, U.S.A. (Received 24 February 1992)
Snmmary--There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37:C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20/~-dihydrocortisol (4-pregnen-11~,17u,20fl,21-tetrol-3-one). The cortisol metabolite, 20fl-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent K~, determined for cortisol metabolism was 12/~M and the observed Vm,xwas 1.4/~mol cortisol/mg protein/min. The majority of the 20fl-reductase activity was found in the soluble (242,000g) fraction of CAM homogenate. 20fl-Reductase activity in chick embryo CAM has not been previously reported.
INTRODUCTION
The chorioallantoic membrane (CAM) of the chick embryo has been used extensively to study angiogenesis [1-7]. In the early stages of embryonic development (days 6-8) the CAM becomes highly vascularized with a dense network of capillaries which extend over the yolk sac. Substances which either promote or inhibit new capillary growth have been identified using this model of natural neovascularization. The CAM is a convenient bioassay system which can be easily accessed for treatment and evaluation, and results are obtained in a short period of time due to the rapid rate of tissue growth [1, 5]. Angiostatic steroids are a specific group of compounds described recently by Folkman and colleagues [2, 4], which inhibit neovascularization on the CAM, independent of hormone activity. Cortisol, a natural glucocorticoid, and its metabolite, tetrahydrocortisol, are among the most active compounds in this group. While angiostatic steroids are potent inhibitors of ne*To whom correspondence should be addressed.
ovascularization on the CAM, no studies have been done to determine if the active agent is the actual steroid molecule applied, or if angiostatic activity is dependent upon metabolism of the steroid by the tissue. The metabolic activity of the early chick embryo CAM for steroids is currently not known. In this report we describe the in vitro metabolism of cortisol by CAM tissue, and compare angiostatic activity of cortisol in the CAM to that of its major metabolite. EXPERIMENTAL Materials
Fertile White Leghorn eggs were purchased from Ideal Poultry Breeding Farms, Inc. (Cameron, TX). The agarose used was Ultrapure, DNA Grade, from Bio-Rad Labs (Richmond, CA). Heparin (calcium salt) from porcine mucosa was purchased from CalBiochem (San Diego, CA). Dimyristoyl phosphatidyl choline was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Unlabeled steroids, cortisol and 20~-dihydrocortisol, were purchased from Steraloids, Inc. (Wilton, NH). Radiolabeled 687
688
LORETTA G. McN^TT et al.
cortisol, ([I,2,6,7-~H]cortisol, 86 Ci/mmol)was purchased from Amersham (Arlington Heights, IL). Solvents were of HPLC grade from Baker Scientific Co. (Phillipsburg, N J). All other chemicals were reagent grade from Sigma Chemical Co. (St Louis, MO).
Cortisol metabolism by CAM tissue Fresh CAM tissue from 6- to 7-day-old embryos was homogenized in 0.25 M sucrose containing 1 mM EDTA (1 ml/gm tissue), and the total volume of homogenate was adjusted to give a protein concentration of 15-20mg/ml. The homogenate was either used without further treatment, or centrifuged at 242,000g to obtain the soluble fraction. Protein concentration was determined by the BCA method (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard. Aliquots of the CAM preparation containing 0.4 mg of protein were added to reaction tubes containing cortisol, NADPH (1 mM), and Tris buffer (50 mM), pH 7.4, in a total volume of 400/al, and incubated at 37°C. The reaction was stopped by adding 600/al of methanol and allowed to stand for 10 min before centrifuging to precipitate proteins and recover the supernatant fluid for analysis by HPLC.
HPLC separations Reverse phase HPLC was run using a C~8 #Bondapak column with 50% methanol in water at a flow rate of 2.0 ml/min. Cyclodextrin HPLC was run using a Cyclobond ITM column with a mobile phase of acetonitrile (50% in water) at a flow rate of 0.8 ml/min. Column et~uent was monitored for u.v. absorption at 214 and 240nm. To monitor [3H]cortisol metabolism, 0.4 ml fractions were collected and added to 4.0ml of OptiFluor scintillation cocktail (Packard Inst. Co., Meriden, CT) for analysis by liquid scintillation spectroscopy using an LKB model 1209 liquid scintillation counter. The major CAM cortisol metabolite was identified by coelution with standard metabolites in both HPLC systems described above.
Petri dishes. Incubation was continued in a humidified incubator in the presence of 3% C02/97% air for an additional 3 days before treatment with angiostatic agents. Suspensions of steroids were prepared in 10% dimyristoyl phosphatidyl choline and mixed with heparin and agarose, to achieve the desired drug dose and 50~g of heparin in 10pl of 1% agarose. Beads of agarose plus drugs, formed by dropping 10/~1 of the suspension onto parafilm and allowing it to gel, were applied to the CAM of 6-day-old embryos. Evaluations of angiostatic response were made by visual inspection 48 h after dosing (day 8). A negative control group treated with agarose beads without drug, and a positive control group treated with a known angiostatic steroid were included in all experiments. A treatment group typically consisted of 10 embryos, and 4-10 treatment groups were evaluated in each experiment. Photographs of representative embryos were made using high contrast black and white film (Polaroid type 5 I). Angiostatic activity was expressed as the percent
(A) 0.6" ~r
0~,.,
$ ~
0.2'
0960-0760/92$5.00+ 0.00 Copyright© 1992PergamonPressLtd
Printedin GreatBritain.All rightsreserved
ANGIOSTATIC CORTISOL
ACTIVITY
IN THE (CAM)
AND
METABOLISM
CHORIOALLANTOIC
OF
THE
CHICK
OF
MEMBRANE
EMBRYO
LORETTAG. McNA'I-r,* DEBORAHLANEand ABBOTF. CLARK Alcon Laboratories, Inc., 6201 South Freeway, Fort Worth, TX 76134, U.S.A. (Received 24 February 1992)
Snmmary--There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37:C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20/~-dihydrocortisol (4-pregnen-11~,17u,20fl,21-tetrol-3-one). The cortisol metabolite, 20fl-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent K~, determined for cortisol metabolism was 12/~M and the observed Vm,xwas 1.4/~mol cortisol/mg protein/min. The majority of the 20fl-reductase activity was found in the soluble (242,000g) fraction of CAM homogenate. 20fl-Reductase activity in chick embryo CAM has not been previously reported.
INTRODUCTION
The chorioallantoic membrane (CAM) of the chick embryo has been used extensively to study angiogenesis [1-7]. In the early stages of embryonic development (days 6-8) the CAM becomes highly vascularized with a dense network of capillaries which extend over the yolk sac. Substances which either promote or inhibit new capillary growth have been identified using this model of natural neovascularization. The CAM is a convenient bioassay system which can be easily accessed for treatment and evaluation, and results are obtained in a short period of time due to the rapid rate of tissue growth [1, 5]. Angiostatic steroids are a specific group of compounds described recently by Folkman and colleagues [2, 4], which inhibit neovascularization on the CAM, independent of hormone activity. Cortisol, a natural glucocorticoid, and its metabolite, tetrahydrocortisol, are among the most active compounds in this group. While angiostatic steroids are potent inhibitors of ne*To whom correspondence should be addressed.
ovascularization on the CAM, no studies have been done to determine if the active agent is the actual steroid molecule applied, or if angiostatic activity is dependent upon metabolism of the steroid by the tissue. The metabolic activity of the early chick embryo CAM for steroids is currently not known. In this report we describe the in vitro metabolism of cortisol by CAM tissue, and compare angiostatic activity of cortisol in the CAM to that of its major metabolite. EXPERIMENTAL Materials
Fertile White Leghorn eggs were purchased from Ideal Poultry Breeding Farms, Inc. (Cameron, TX). The agarose used was Ultrapure, DNA Grade, from Bio-Rad Labs (Richmond, CA). Heparin (calcium salt) from porcine mucosa was purchased from CalBiochem (San Diego, CA). Dimyristoyl phosphatidyl choline was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Unlabeled steroids, cortisol and 20~-dihydrocortisol, were purchased from Steraloids, Inc. (Wilton, NH). Radiolabeled 687
688
LORETTA G. McN^TT et al.
cortisol, ([I,2,6,7-~H]cortisol, 86 Ci/mmol)was purchased from Amersham (Arlington Heights, IL). Solvents were of HPLC grade from Baker Scientific Co. (Phillipsburg, N J). All other chemicals were reagent grade from Sigma Chemical Co. (St Louis, MO).
Cortisol metabolism by CAM tissue Fresh CAM tissue from 6- to 7-day-old embryos was homogenized in 0.25 M sucrose containing 1 mM EDTA (1 ml/gm tissue), and the total volume of homogenate was adjusted to give a protein concentration of 15-20mg/ml. The homogenate was either used without further treatment, or centrifuged at 242,000g to obtain the soluble fraction. Protein concentration was determined by the BCA method (Pierce Chemical Co., Rockford, IL) with bovine serum albumin as a standard. Aliquots of the CAM preparation containing 0.4 mg of protein were added to reaction tubes containing cortisol, NADPH (1 mM), and Tris buffer (50 mM), pH 7.4, in a total volume of 400/al, and incubated at 37°C. The reaction was stopped by adding 600/al of methanol and allowed to stand for 10 min before centrifuging to precipitate proteins and recover the supernatant fluid for analysis by HPLC.
HPLC separations Reverse phase HPLC was run using a C~8 #Bondapak column with 50% methanol in water at a flow rate of 2.0 ml/min. Cyclodextrin HPLC was run using a Cyclobond ITM column with a mobile phase of acetonitrile (50% in water) at a flow rate of 0.8 ml/min. Column et~uent was monitored for u.v. absorption at 214 and 240nm. To monitor [3H]cortisol metabolism, 0.4 ml fractions were collected and added to 4.0ml of OptiFluor scintillation cocktail (Packard Inst. Co., Meriden, CT) for analysis by liquid scintillation spectroscopy using an LKB model 1209 liquid scintillation counter. The major CAM cortisol metabolite was identified by coelution with standard metabolites in both HPLC systems described above.
Petri dishes. Incubation was continued in a humidified incubator in the presence of 3% C02/97% air for an additional 3 days before treatment with angiostatic agents. Suspensions of steroids were prepared in 10% dimyristoyl phosphatidyl choline and mixed with heparin and agarose, to achieve the desired drug dose and 50~g of heparin in 10pl of 1% agarose. Beads of agarose plus drugs, formed by dropping 10/~1 of the suspension onto parafilm and allowing it to gel, were applied to the CAM of 6-day-old embryos. Evaluations of angiostatic response were made by visual inspection 48 h after dosing (day 8). A negative control group treated with agarose beads without drug, and a positive control group treated with a known angiostatic steroid were included in all experiments. A treatment group typically consisted of 10 embryos, and 4-10 treatment groups were evaluated in each experiment. Photographs of representative embryos were made using high contrast black and white film (Polaroid type 5 I). Angiostatic activity was expressed as the percent
(A) 0.6" ~r
0~,.,
$ ~
0.2'
