EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

56, 1-19 (1992)

Characterization and Applications Angiogenesis System’

of the Disc

JOE KOWALSKI,HELEN H. KWAN,* STAVROS D. PRIONAS,? ANTHONY C. ALLISON, AND LUIS F. FAJARDO*,' institute of Immunology and Biological Sciences, Syntex Research, Palo Alto, California 94304; and Departments of *Pathology and FRadiation Oncology, Stanford University School of Medicine, and Veterans Affairs Medical Center, Palo Alto, California 94304 Received July 3, 1991, and in revised form October 28, 1991 A model to study microvascular proliferation, the Disc Angiogenesis System (DAS), consists of a synthetic foam disc implanted subcutaneously in experimental animals. After a period of growth, usually 7 to 21 days, the disc is removed. Planar sections are used to measure and characterize the growth. Microvessels grow centripetally into the disc, together with fibroblasts. Concentric growth zones have been defined by light and electron microscopy. Moderate growth occurs spontaneously and is accelerated by angiogenic stimulants placed in the center of the disc. Morphometric analyses have shown that vessel growth is directly proportional to total fibrovascular growth, so the former can be quantified by procedures measuring the latter. These include manual projection of sections and computer-assisted digital image analysis, which is recommended for routine use. The proliieration of endothelial and other cells is determined by incorporation of tritiated thymidine, using scintillation counting and autoradiography. Using the DAS, well-established angiogenie agents such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and prostaglandin E, were found to increase proliferation of endothelial cells (EC) and microvessels. Heparin augmented the effect of bFGF. When used by itself heparin increased angiogenesis but not EC proliferation, in keeping with in vitro observations indicating that it stimulates migration but not proliferation of EC. Locally applied hyperthermia and ionizing radiation decreased angiogenesis, even when applied after the angiogenic stimulus. Systemic prostaglandin synthetase inhibitors antagonized the angiogenic effects of bFGF and EGF, in accordance with a postulated role of prostaglandins in the transduction of proliferative signals in microvascular EC. The DAS is easy to assemble and implant in small animals, including mice, which tolerate it well. Hence multiple discs can be used for each time or dose point, which allows reproducible measurements of vascular growth and increases statistical accuracy. Another advantage of the system is the capability of discriminating between proliferation and migration of EC and tibroblasts. The DAS can be used to test putative agonists or antagonists of angiogenesis. More generally, the DAS provides a model of wound healing, either uncomplicated or complicated by intlammation, and of angiogenic responses to solid tumors. 8 wz Academic press, h.

INTRODUCTION In adult vertebrates, angiogenesis is a complex process involving a cascade of cellular events that includes production of proteases, migration and proliferation of endothelial cells (EC), formation of a vessel lumen, synthesis of new basal lamina, and ultimately blood flow (Folkman, 1982; Furcht, 1986). It plays a critical role in several pathological conditions, including chronic infIammation, diabetic retinopathy, and tumor growth as well as in physiological responses such as wound repair and cyclical endometrial proliferation. Both the promotion of an’ Supported by Syntex Research and Department of Veterans Affairs Research Funds (MRIS 273501). * To whom reprint requests should be addressed at Department of Pathology (113), Veterans Affairs Medical Center, Palo Alto, CA 94304.

1 00144800/92 $3.00 Copyright 8 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

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giogenesis and its inhibition have potentially important therapeutic applications. Therefore the isolation of specific angiogenic factors and definition of their mode of action have been the object of much study (Allison and Kowalski, 1989; Auerbath, 1981; Buckley and Woodward, 1985; Cotran, 1987; Folkman, 1990; Schor and Schor, 1983; Zetter, 1988). Several methods to study angiogenesis have been proposed, each with its own particular advantages and disadvantages. The following is a detailed description of a model-the Disc Angiogenesis System: DAS-that we introduced several years ago (Fajardo ef al., 1988a). Its design has been improved and we have devised easier and more accurate procedures to measure the vascular growth. The sequence of morphologic and proliferative changes occurring in the disc has now been established and is presented here, together with quantitative base-line data and examples of the applications of the DAS. This description should provide important (and previously unavailable) information for the various laboratories currently using the DAS. DESCRIPTION

OF THE DAS

The DAS was based on our initial observation of vascular growth into subcutaneously implanted sponges (Fajardo et al., 1988a). Several versions of the system have been tested during more than 3 years. The DAS currently used consists of a disc of polyvinyl alcohol sponge (Kanebo, PVA, Rippey Co., Santa Clara, CA) 13 mm in diameter and 2-mm thick. The flat sides of the disc are covered with cell-impermeable filters (0.45 Frn, HAWP 013, Millipore) sealed by No. 1 Millipore glue (XX70 000 00, Millipore) (Fig. 1). This leaves only the external rim (a 2-mm wide band) for penetration or exit of cells. When indicated, and prior to placement of the filters, a 3 mm in diameter hole is bored in the center of the disc. A pellet of the same sponge material 2 mm in diameter, containing an angiogenic agonist or antagonist is coated with acetate copolymer (Elvax, DuPont; distributed by Chemcentral Corp., Chicago, IL) and placed in the central hole. Each pellet of this size can hold up to 20 pl of the material to be tested. The angiogenic stimulants that we have used most often are recombinant human epidermal growth factor (EGF; usually 20 kg per disc) and recombinant human basic fibroblast growth factor (bFGF; usually 20 kg per disc). Preferably the preparation of the disc is made from sterile materials and under sterile conditions, within a laminar-flow hood. Alternatively nonsterile discs can be sterilized by a variety of methods. However, care should be exercised to avoid physical or chemical damage to the sponge, or to the angiogenic or inhibitory agents already in the disc. For instance, the sponge tolerates dry heat up to 120°C but it can be damaged by boiling water. The mouse is a convenient host animal because of its well-defined genetics. Also several recombinant mouse growth factors are available. Its small size is economically advantageous, especially when testing substances are in short supply. We have used mice of either sex, most often of the Swiss Webster or BALB/c strains, approximately 8 weeks old and weighing 20-30 g. Other investigators in various laboratories have used the DAS in rats or rabbits (personal communications). Consistency in the handling of the animals and placement of the discs has been found to be critical to the success of a given experiment. However, good fibrovascular growth occurs whether the disc is placed, subcutaneously, in the abdomen or thorax. The mouse is anesthetized by an intraperitoneal injection of ketamine

ANGIOGENESIS

MODEL

FIG. 1. Diagram indicating sequential disc assembly, implantation, removal, embedding, sectioning, and analysis (quantitative and qualitative). See text for details of each.

hydrochloride (50 mglkg), xylazine (5 mg/kg), and acepromazine maleate (1 mg/ kg). The shaved skin surface is bathed in 70% ethanol, and a 1.5cm-long incision into the subcutis is made at least 1 cm away from the desired location of the DAS. Blunt dissection is used to produce a tunnel toward the site of implantation. Phosphate-buffered saline (PBS) is dripped into the area with a Pasteur pipette. Holding it gently with forceps, the PBS-moistened disc is then inserted through the wound and into the tunnel up to the desired site, and the skin wound is closed with three to four metal clips. The animals are then housed as usual, with standard chow and water ad libitum, during the period of the experiment (7 to 30 days, most often 14 days). Mice tolerate well the implants and do not scratch incisions made on the dorsum or the flank. Uptake of tritiated thymidine (3HTdR) is used to evaluate DNA synthesis and, therefore, proliferation of endothelial and other cells (Hobson and Denekamp, 1984). Total incorporation of label is measured by scintillation counting while autoradiography identifies the different cell types synthesizing DNA. To label cells in the disc, we have injected into mice, intraperitoneally [methyl-3H]thymidine (sp. act., 6.7 Ci/mmole, DuPont: 66 &i/25 g body wt) at 24, 18, and 12 hr before sacrifice (total = 200 &i), or delivered it continuously by minipumps (Alza Co., Palo Alto, CA) implanted subcutaneously for 7 or 14 days. To aid in recognizing vessels by light microscopy, an injection of Luconyl blue (BASF; distributed by Wyandotte Co., Hotland, MI; 0.4 ml of a 40% solution in PBS) (Reinhold et al., 1983) is given intracardially to the mouse 15 min prior to euthanasia. The latter is carried out with a large i.p. dose of anesthetic, followed

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by quick cervical dislocation. A careful incision is made in the skin overlaying the implanted DAS; the disc is dissected gently from the surrounding tissues and removed. After removal, one of the filters is carefully dissected off with a sharp blade to permit effective fixative penetration and dehydration. The disc is placed in 10% neutral formalin for 48-72 hr and then embedded flat in paraffin, so the initial sections will be taken from the uncovered side of the disc. Multiple planar sections, 6-pm thick, are obtained (Fig. 1): for light microscopy and measurement of radial growth (hematoxylin-eosin); for measurement of total area growth (toluidine blue); for autoradiography; and for scintillation counting, etc. CHARACTERISTICS

OF VASCULAR

GROWTH

IN THE DAS

Light Microscopy

The growth into the disc is composed of blood vessels and surrounding stroma (Figs. 1 and 2) and is usually asymmetric. This asymmetry may be related to the density of mother vessels (venules) in the surrounding host tissue, from which the vessel sprouts are derived. The stroma contains libroblasts, macrophages, neutrophils, and occasional lymphocytes, in that order of frequency (Fig. 3). The various proportions of these elements vary considerably between the inner zone of the disc and the outer zone (as divided by an arbitrary circular line into two bands). Some time-dependent variation is observed, but this is not substantial. There is no significant difference in the proportion of cell types between discs with spontaneous growth (i.e., occurring without stimulant) and discs in which growth has been stimulated by endothelial growth factors. Neither the 14- nor the 27-day samples contain mast cells within the angiogenesis disc. By using metachromatic stains a moderate number of mast cells can be demonstrated in the tissues of the host immediately around the disc, at Day 20 and beyond. Mast cells may have been present earlier and become degranulated. Throughout the entire area of growth the majority of cells are fibroblasts; the second most common cells are endothelial; and other cells (neutrophils, macrophages etc.) comprise the rest. By 14 days of growth stimulated by 20 pg of EGF the proportions of these three categories of cells are 53,25, and 22%, respectively. However, in the outer zone of the disc, the cellularity is high, while in the inner zone it is low (Figs. 3A and 3B). Although nonendothelial cells predominate in both zones (67-81% at Day 14 of spontaneous growth), the proportion of EC is higher in the inner zone than in the outer zone (33% vs 19% at 14 days of spontaneous growth). The space occupied by the various elements has been determined by point counting with a grid in standard histology sections (Chalkley, 1943). The sponge trabeculae occupy a constant proportion of the disc (around 30%). At 14 days of EGF-stimulated growth, fibroblasts comprise 18% and blood vessels only 5%; a large portion of space (>46%) is not occupied by vessels, cells, or trabeculae. The area of the disc occupied by blood vessels is a consistent percentage of the total growth area regardless of the size of the latter. In other words, as the area grows from Day 9 to Day 23, the portion occupied by blood vessels grows parallel to the growth of the total fibrovascular area. Hence, the measurement of the total area is an accurate index of the size of the blood vessel area (Fig. 4). Ultrastructure

Electron microscopy

samples have been obtained at 14 and 27 days from discs

ANGIOGENESIS

MODEL

FIG. 2. Section of DAS showing empty polyvinyl sponge in the upper one-third of the photograph (inner portion of disc). The lower two-thirds (outer, or peripheral portion of disc) contain vessels and tibroblasts among the sponge trabeculae. The rim of the disc is at the bottom. Disc stimulated by 20 pg of EGF, 14 days after implantation. H&E x50.

stimulated by bFGF (20 pg). In the 1Cday discs, there are two different growth zones: outer (peripheral) and inner (central) which can be separated by dividing the growth area into two equally wide bands. In the 27-day sample, which has a wider area of growth, there are three different, almost equally wide bands: outer, middle, and inner. Vessels. Outer samples show well-formed capillaries and sinusoids with flat or low-ovoid endothelial cells connected by junctional complexes (Fig. 5). Vessels are often wide and have irregular outlines, each with an ample lumen. EC have more often flat rather than oval nuclei. Their cytoplasm contains a few prominent profiles of rough endoplasmic reticulum (RER), inconspicuous small mitochondria, and many polyribosomes. Pinocytotic vesicles are present in practically all EC but are not abundant in any; a few EC have lysosomes. Most vessels have a well-defined, thin basement membrane. However in a few vessels, usually in wide sinusoids, the basement membrane is not detectable. The lumenal border of the

KOWALSKI

ET AL.

ANGIOGENESIS

MODEL

7

TIME (days)

FIG. 4. Vessel growth is directly proportional to total fibrovascular growth. As the latter increases continuously (upper line) during the period of observation, the proportion of the growth area occupied by blood vessels (lower line) remains constant. Total growth area was measured by computer-assisted digital image analysis and vascular area by point counting (see text). Growth stimulated by 20 ug of EGF. Bars: standard error of means.

EC is generally smooth, but some cells have irregular, multiple, or single projections of cytoplasm. Occasional mitotic figures are seen in EC. In no vessel are there recognizable smooth myocytes, i.e., there are no venules or arterioles (Bruns and Palade, 1968). However, most of the vessels are partially surrounded by flat or spindle-shaped cells closely apposed to the outer surface of the basement membrane and occasionally surrounded by the basement membrane (Fig. 6). These pericytes (Cracker et al., 1970) have prominent RER and elongated mitochondria, and some small bundles of microfilaments. Samples from the midzone have similar vessels to those of the outer zone, although the basement membrane is generally more prominent in the mid-zone. A few vessels contain aggregates of platelets, without fibrin. The inner zone vessels are of two types: One type has the morphology of the outer and mid-zone vessels as described above. Nearly each vessel of this type has a pericyte, sometimes completely surrounding the vessel (Cracker et al., 1970). The other type consists of small vessels with narrow lumina and cuboidal or pyramidal EC (Fig. 6). The latter appear to be the least differentiated vessels. All of these have well-defined basement membranes; some are surrounded by pericytes while others are not. In the inner zone, it is possible to find a progression from tiny capillary lumina, appearing as slits within individual EC (Fig. 7), through capillaries with a small lumen lined by a few tall cells, to well-formed vessels with highly developed lumina, lined by flat EC. Stroma. The outer zone usually contains a large amount of collagen continuous with that of the host tissue outside the disc. Fibroblasts are numerous and mac-

FIG. 3. Sections of DAS. (A) The inner, leading zone of growth has a sparse population of tibroblasts (spindle-shaped but may be oval), little collagen, and a few leukocytes. Capillaries, such as the dark Y-shaped one in the center are often slender and oriented radially. The large lobulated masses of gray material with “bubbles” are the sponge trabeculae. Notice some erythrocytes in left lower comer. x460. (B) The outer (peripheral, old) zone of growth has a dense population of tibroblasts and the vessels are often broader than in the inner zone. The vessel diagonally oriented in the center contains Luconyl blue. x480. Both H&E.

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detail of a transversely sectioned outer zone capillary. In the center, beFIG. 5. Ultrastructural tween arrows, is an endothelial cell junction containing three dense zones suggestive of maculae adherentia, and a zonula occludens. The basal lamina (tip of big arrow) is delicate and characteristically fuzzy. Polyribosomes are prominent in the left cell and mitochondria in the right. A group of pinocytotic vesicles is near right edge of lumen. The disc contained 20 pg basic FGF. Uranyl acetate and lead citrate. ~40,600.

rophages are easy to find. The latter, characteristically, are large cells with numerous round or oval mitochondria, prominent RER, and many ruffles of the plasma membrane. These cells contain moderately dense membrane-bound material in the form of round or star-shaped inclusions. This material is probably derived from the matrix of the sponge. In fact some macrophages are wrapped around the sponge trabeculae; these are generally multinucleated. Loose red cells are common and there are some lymphocytes and neutrophils. The densely cellular and collagen-rich stroma seen in the outer zone contrasts with the stroma-poor mid-zone: only some red cells and occasional fibroblasts are seen among the microvessels which, as compared with the outer and inner zones, are few and far apart. The inner zone has a greater concentration of vessels than the mid-zone, but considerably less stromal elements than the outer zone. Macrophages, however, are easy to find as well as red cells and some neutrophils.

ANGIOGENESIS

MODEL

9

FIG. 6. Vessel from the inner zone of growth. Endothelial cells are pyramidal, with an outer base invested by a well-defined basement membrane. Lumen is small and contains a minute portion of a tangentially sectioned red cell. Most of this vessel is encircled by a pericyte (right) with a flattened nucleus and prominent rough endoplasmic reticulum. This cell is also covered by a basement membrane. The disc contained 20 ug basic FGF. Uranyl acetate and lead citrate. X 12,600.

Interestingly enough, fine collagen fibrils are often seen around the vessels, far less dense than in the outer zone but more common than in the mid-zone. The main difference between the 1Cday and 27-day specimens is the larger growth area of the latter.

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ANGIOGENESIS

QUANTIFICATION

11

MODEL

OF ANGIOGENESIS

IN THE DAS

One of the advantages of the DAS is the ability to measure vascular growth. Direct measurement of the blood vessels can be performed by various methods, including point counting on histologic sections (Haynes, 1964), determination of intravascular volume, e.g., with radioactive isotopes (Mahadevan et al., 1989), etc. Such methods are tedious and impractical when examining a large number of discs. Thus, indirect, simpler procedures have been devised. One such method consists of measuring the distance between the blood vessel closest to the center of the disc and the peripheral edge of the disc (Fig. 1). The innermost blood vessel is identified and the radial distance between the leading tip of such a vessel and the rim of the disc is measured in centimeters, on a section projected (by a Bausch and Lomb microprojector) at 100x magnification onto a horizontal surface. We have named this end-point the “centripetal vessel growth” (CVG). The CVG is directly proportional to the total vessel growth and therefore it is a reliable indirect measure of such growth (Fajardo et al., 1988a,b). By comparing the total growth area with the area occupied by blood vessels, we have shown that the latter is consistently proportional to the former (Fig. 4). Therefore, the measurement of the total growth area is in fact an accurate, indirect measurement of the area occupied by blood vessels. A method of determining the total growth area is to measure the radial fibro-vascular growth along two perpendicular diameters (four radii) using the above-mentioned projection apparatus (Fig. 1). The average value of the four measurements is obtained for each disc. A precise system of measuring total growth area uses paraffin sections (6-km thick) that have been uniformly stained with toluidine blue to obtain high contrast (Fig. 1). The toluidine blue stains uniformly the area of growth and does not stain the sponge trabeculae. The sections (mounted on slides without coverslips) are placed on a microscope fitted with a computer-assisted digital image analysis system (Image Measure/IP Ver. 4.0) which measures the entire area of growth automatically in pixels. Threshold values are standardized for a particular experiment, and conversion to metric area (mm*) is accomplished through the use of an ocular grid. We recommend this computer-assisted method of analysis for reproducible and rapid measurements. Differential counting of the various cells that comprise the fibrovascular growth is performed with microscope retitles. The relative proportions of various cell types, as well as the absolute numbers of the cells per given area, are established by differential counting within standard-size areas of the various zones of growth in the DAS (Fig. 1). The space occupied by vessels and other elements is determined by using the intersection points of microscope grids. Such procedures, which are time-consuming, were used initially to analyze the biology of fibrovas-

FIG. 7. Three early stages in the development of capillaries, in the inner zone. (A) An endothelial cell (EC) encircling a thin, sigmoid-shaped lumen to the right of the nucleus. Two cell junctions are present to the right of the lumen. This EC may be at the tip of a vascular sprout. (B) An EC with a wider lumen than in (A). No cell junction is seen at this level. (C) A capillary with a wider lumen and thinner wall containing at least one cell junction. That the latter capillary level had already blood flow is evident by the presence of a red cell. Each of these vessels has a recognizable basement membrane, and each (when examined at higher magnification) exhibits numerous pinocytotic vesicles. The disc contained 20 kg basic FGF. Uranyl acetate and lead citrate. All X 10,600.

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cular growth and to validate the more often used quantitative methods described in the previous paragraphs. For scintillation counting, 10 serial 6 km-thick sections are subjected to alkali solubilization and their B radiation activity is determined in a scintillation counter. The values obtained represent the total amount of thymidine incorporation, i.e., DNA synthesis of all cells in the sample. Autoradiographs of 6 km-thick paraffin sections are prepared by dipping the slide-mounted sections in photographic emulsion (NTB-2 emulsion, Eastman Kodak Corp.). These slides are held at 4°C for 35 days and developed. The developed slides are counterstained with hematoxylin and eosin. In these autoradiographic preparations differential counting of labeled vs unlabeled cells has been performed using standard size microscopic fields: two in the outer zone and two in the inner zone of each disc. Cells containing five or more grains have been considered as labeled (in fact, after a 5-week exposure, the great majority of labeled cells contain more than 10 grains per cell. Background is less than one grain per 100 pm*) (Fig. 8). From overall incorporation of tritiated thymidine into DNA, as determined by scintillation counting, and autoradiographic studies of the ratio of incorporation into endothelial cells as compared with other cell types, the rate of DNA synthesis in endothelial cells can be calculated. Raw data are analyzed in an IBM-PC/AT computer using the RSI statistical analysis software. Having obtained means, standard errors, and standard deviations for each group of discs, comparison of experimental and control groups are carried out using Student’s t test. NORMAL

GROWTH

We have studied sequentially the fibrovascular growth in the DAS. The typical growth curve induced by EGF is shown in Fig. 9. The total librovascular growth

FIG. 8. Autoradiograph of paraffin section from DAS stimulated by 20 pg of bFGF. The diagonally oriented capillary in the center has at least two endothelial cells labeled by 3HTdR. The labeled cells outside of the capillary are fibroblasts. Inner zone of growth. Counterstained by H&E. x460.

ANGIOGENESIS

MODEL

13

1 50

--I

FIG. 9. Total growth area (measured by digital image analysis) is compared with total cell proliferation (as determined by 3HTdR incorporation over 24 hr) in discs containing 20 pg of EGF. While total growth area is continuously increasing, ‘HTdR incorporation decreases from Day 14 on. Each data point represents the average of 5-10 samples. Bars: standard error of the means,

area increases continuously, with some plateau between 14 and 17 days. The total incorporation of 3HTdR (also shown in Fig. 9) increases exponentially, reaching maximum at Day 14, and then declines continuously to Day 23. This decline in the number of cells undergoing DNA synthesis, in the face of continuing tissue growth, indicates that beyond Day 14 cell migration makes a proportionately greater contribution than cell proliferation. From multiple 3HTdR autoradiography experiments it is cJear that most of the cells synthesizing DNA are located in the inner zone, as opposed to the outer zone. For example, on the 14th day of spontaneous growth 49% of the EC in the inner zone are labeled (vs 7% in the outer zone). The same is true for fibroblasts (35% vs 6%). APPLICATIONS

OF THE DAS

This system has been used to test the effects of putative agonists and antagonists of angiogenesis, pharmacological and otherwise. We have performed multiple experiments using the DAS for this purpose, some of which are described below. Initially we tested the effects of known angiogenic agonists. Epidermal growth factor (EGF) or prostaglandin E, (PGE,) was placed in the center of the discs and shown to increase fibrovascular growth. These agonists also increased DNA synthesis by endothelial cells. Heparin, used by itself, increased the area of fibrovascular growth without increasing DNA synthesis, whereas heparin plus bFGF increased DNA synthesis as well as the growth area (Fig. 10). The DAS was then applied to study the effects of postulated inhibitors of angiogenesis, either local (hyperthermia and X-radiation) or systemic (glucocorticoids and cyclooxygenase inhibitors). The hypothesis that hyperthermia-as used in cancer therapy-interferes with angiogenesis was based on our in vitro observation that capillary EC are sensitive to hyperthermia, which is lethal for EC in a dose-dependent fashion (Fajardo et al., 1985). The experimental dose re-

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n 3HTdR (cpm) •l GROWTH AREA

C

HEP

FGF

(mm*)

FGFlHEP

Heparin promotes angiogenesis mainly by stimulating cell migration. Comparison of total growth area (hatched bars) with cell proliferation (expressed as 3HTdR incorporation; solid bars) indicates that little or no cell proliferation contributes to total growth stimulated by heparin alone. When bFGF is added, both growth and proliferation are increased. C, Spontaneous growth; HEP, porcine heparin (Sigma, 50 p,g per disc); FGF, basic tibroblast growth factor (20 pg per disc); FGF/ HEP, 20 pg FGF and 50 pg HEP per disc. A total of 200 pCi of ‘HTdR was infused subcutaneously over 14 days by an Alza osmotic minipump. Both growth and cell proliferation are expressed as percentage of control values. At1 samples analyzed at 14 days of growth. Standard errors of means are indicated. Statistically significant differences (P < 0.05) in growth area were observed in the following groups: HEP vs C, FGF vs C, FGFlHEP vs C, and FGFlHEP vs HEP; and FGF/HEP vs C in 3HTdR incorporation. FIG.

10.

quired to inhibit vascular growth by 50% was 42.O”C for 30 min. The results left no doubt that hyperthermia, within the dose range used in clinical cancer therapy, inhibits angiogenesis (Fajardo et al., 1988b). Hence, at least some of the antitumoral effects of hyperthermia are the result of interference with tumor microcirculation . In another study (Prionas et al., 1990) the effects of X-radiation on angiogenesis were analyzed. A dose-response relationship was observed when X-radiation was administered on Day 11 after implantation and the disc was removed on Day 20 (Prionas et al., 1990). The dose of X-irradiation required to cause a 50% reduction in growth area was approximately 10 Gy. These and previous observations point to endothelial cells as important targets of ionizing radiation in the stroma, especially during the period of active proliferation of these cells induced by growth factors. Effects of systemically administered small molecular inhibitors of angiogenesis were also studied. Glucocorticoids are potent inhibitors of endothelial cell proliferation in culture and of angiogenesis in vivo (Folkman et al., 1983; Gross et al., 1981; Ingber et al., 1986). In the DAS glucocorticoids were found to be highly effective in counteracting the angiogenic effects of EGF and bFGF. For reasons outlined in the discussion we postulated that E- and I-type prostaglandins might be transducers of the angiogenic effects of bFGF, which implied that a cyclooxygenase inhibitor might block the in vivo angiogenic effect of bFGF. We used ketorolac (5-benzoyl-2,3-dihydro-lN-pyrrolizine-l-carboxylic acid tris (hydroxymethyl)aminomethane salt), a potent cyclooxygenase inhibitor (Rooks et al., 1985). Using the DAS model, this was found to be the case: ketorolac inhibited

ANGIOGENESIS

15

MODEL

bFGF-stimulated angiogenesis, presumably because it interfered with the synthesis of new PGE; however, it did not affect angiogenesis stimulated by already synthesized PGE (Fig. 11). ADVANTAGES

AND DISADVANTAGES

OF THE DAS

Previously described angiogenesis models include the rabbit ear chamber (Sandison, 1924), the rat air pouch (Reinhold et al., 1979), the hamster cheek pouch (Warren and Shubik, 1966), the chick chorioallantoic membrane (Alfthan, 1956), and the rabbit or rat cornea assay (Gimbrone et al., 1974; Proia et al., 1988). Wound healing chambers have been made of materials such as wire mesh (Hunt et al., 1967), or porous polytetrafluorethylene tubes filled with collagen (Sprugel et al., 1987). Alginate (Plunkett and Hailey, 1990), fibrin (Dvorak et al., 1987), and synthetic polymers (Andrade er al., 1987; Mahadevan et al., 1989) have also been used as matrices for vascular growth. Each of these angiogenesis models has its own advantages and disadvantages, and each one has an application for which it is best suited. For instance, observation of living individual capillaries as they form and become patent is best done in the rabbit ear chamber (Sandison, 1924) or its successors, the rat pouch (Reinhold et al., 1979), the hamster chamber (Greenblatt and Shubik, 1968), etc. Sequential recording of the growth of multiple capillaries into a biological material is best done in the cornea (of rabbits, rats, etc.) (Gimbrone et al., 1974). The measurement of vascular growth is best performed in the DAS.

50

7

45 -40 -:35

_-

E ~30.. B < 25 -3 20 &

15

t

10 5 F

0 I

C

PGE,

PGE, KETO

FGF

FGF KETO

FIG. Il. Effect of cyclooxygenase inhibitor (ketorolac) on angiogenesis. In vitro studies have indicated that capillary endothelial cell proliferation stimulated by fibroblast growth factor requires the presence of prostaglandins. As shown here in the DAS, the same occurs in viva: ketorolac (4 mgikg daily by gavage) inhibits the M-day angiogenesis stimulated by bFGF (20 ug; last two bars), presumably because it interferes with the synthesis of new PGE. However, ketorolac does not have an effect on angiogenesis stimulated by already synthesized PGE (PGE,; 75 kg.in the center of the DAS) as shown in the second and third bars. The control DAS in the first bar contained no agonist or antagonist and thus indicates spontaneous growth. C, Control; PGE,, prostaglandin E,; FGF, basic fibroblast growth factor; KETO, ketorolac. Standard errors of means are indicated.

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Advantages of the DAS The disc consists of easily obtainable and inexpensive materials. Many discs can be used in one experiment. Being small, it is simple to implant in various animal species, particularly rodents. The discomfort associated with the corneal assays does not occur with the DAS. It is well tolerated and causes little or no skin inflammatory reaction that could interfere with the process of angiogenesis. The animals do not scratch the incision wound or the site of implant. The DAS is a mammalian system, unlike the chorioallantoic membrane, and therefore more relevant to human physiology and pathology. In the DAS there is always a moderate, well-characterized, and measurable spontaneous vascular growth, while in other systems, there is little or no spontaneous vascular growth. Therefore in the DAS one can not only test angiogenic agonists but also antagonists, the latter by measuring the decrease in the spontaneous growth. Unlike in some angiogenesis models where microvessels grow with little or no stroma, in the DAS vessels and stromal elements grow concomitantly. The latter replicates the mesenchymal proliferation that occurs in physiologic (e.g., proliferative endometrium, wound healing) and in pathologic conditions (e.g., chronic infhunmation, neoplasms), characterized by angiogenesis. The most important advantage of the DAS is the fact that vascular growth can be quantified easily and reproducibly. This is not so in most other systems. For instance, measurement of vessel growth in the cornea involves complex and lengthy procedures (Proia et al., 1988). Discriminating between new and preexisting vessels in the hamster pouch can be difftcult, while in the DAS all vessels are new by definition. The entire circumference of the DAS is available in a section, and all cellular elements can be identified by appropriate stains. In the DAS multiple vessels can be studied and measured and multiple discs can be used for each point; in the systems designed for visual monitoring of living vessels (e.g., rabbit ear chamber), only a few vessels can be measured and, because of cost, there may not be enough vessels, or animals, to obtain statistical significance. Finally, the growth curves and the relative proportions of vessels and fibroblasts are now well established for the DAS. Disadvantages of the DAS The system is not designed for, nor does it allow, progressive observations of living blood vessels. External and gross inspection cannot be performed. Each disc provides information for only one point in time. Histologic embedding and staining are required. DISCUSSION The DAS was devised as a system for quantification of microvascular growth. The sequential events occurring in this model are now well characterized and reproducible. For instance, proliferation of EC in the newly forming capillaries is confined to the leading, inner zone of growth. The ratio of microvessels to total growth area remains constant at different times, so that measurements of total fibrovascular growth provide a valid index of angiogenesis. Total fibrovascular growth is easily and quantitatively measured by computer-assisted image analysis of toluidine blue-stained sections, which is recommended for routine use. Proliferation of EC can be assessed independently of migration by tritiated thymidine labeling. Counting 3HTdR in the whole disc provides information on overall DNA

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synthesis, while autoradiography discriminates between DNA synthesis in endothelial cells and other cell types. The two methods provide complementary information. The DAS has been used to assess the angiogenic and anti-angiogenic activities of various agents. The principal motivation for studying angiogenesis has been to elucidate the process as required for growth of solid tumors. Among the defined angiogenic factors produced by tumor cells, which have been obtained by recombinant DNA technology, are EGF, transforming growth factor-a, (TGFa, which binds to EGF receptors) (Schreiber et al., 1986), and basic and acidic FGF (Maciag et al., 1984; Montesano et al., 1986). Two of these, recombinant EGF and bFGF, have been tested in the DAS and found to increase EC proliferation and angiogenesis. X-radiation and hyperthermia both inhibit angiogenesis in the DAS, and this may be one of the ways in which they exert anti-tumor effects. Angiogenesis is also a feature of chronic inflammatory reactions. The proliferative synovial tissue which erodes cartilage and bone in patients with rheumatoid arthritis contains mitotically active EC (Young et al., 1986). One of the ways in which methotrexate exerts antirheumatic activity is believed to be the inhibition of endothelial cell proliferation (Hirata et al., 1989). This may also be true of cyclooxygenase inhibitors: elsewhere (Allison and Kowalski, 1989) we have presented evidence that E-type prostaglandins and adenylate cyclase activation are essential components of transduction signals by which growth factors induce proliferation of microvascular EC. As shown in Fig. 11, systemic administration of a cyclooxygenase inhibitor (ketorolac) negates the angiogenic effect of bFGF, but not that of prostaglandin El. These observations are also relevant to the treatment of granulation tissue reactions associated with continued exposure to microbial products. A common example is chronic inflammatory periodontal disease, a response to gingival bacterial products (Page, 1986). Newly formed blood vessels in the granulation tissue of the gums are fragile and bleed easily. Topically applied ketorolac inhibits the periodontal granulation tissue reaction in experimental animals. Ketorolac topically applied to the eyes of rats was also found to inhibit silver-nitrate-induced cornea1 neovascularization (Rooks et al., 1985). The examples given illustrate applications of the DAS for analyzing angiogenesis associated with tumor cell growth and inflammatory reactions. In addition to the above type of studies the DAS can be used for other purposes as follows: (1) To study fibrogenic agents or inhibitors, the system allows separate quantification of the migration and proliferation of fibroblasts and the amount of collagen synthesized, e.g., by extraction of hydroxyproline from the disc. Types and subtypes of collagen may also be determined immunologically. (2) To test in viva the relative importance of different stromal components in EC proliferation and migration, as suggested by in vitro studies (Madri et al., 1983), the sponge of the DAS can be coated before implantation with fibronectin, vitronectin, proteoglycan, various types of collagen, and other connective tissue components. Results already obtained with heparin are instructive in this respect. (3) To determine the angiogenic potential of cells, especially neoplastic cells, we are currently testing tumor cells contained in agar pellets. This will be followed by the use of tumor spheroids (Sutherland, 1988). There are multiple additional potential applications of the Disc Angiogenesis System, too long to list here. We hope that other investigators continue to use it with satisfactory results.

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ACKNOWLEDGMENT We appreciate the assistance of Donna L. Buckley.

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