Tumor Angiogenesis

TUMOR ANG IOGENESIS Judah Folkman Department of Surgery. Children's Hospital Medical Center and Departments of Surgery and Anatomy, Harvard Medical School. Boston. Massachusetts

I. Introduction . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . ,

11. Methods for Studying Angiogenesis A. The Corneal Micropocket . . . . .

111.

IV. V. VI. VII. VIII.

IX.

X. XI. XII.

B. Sustained-Release Polymer Implants C. Chick Embryo Chorioallantoic D. Cloned Capillary Endothelial Cells A Capillary Grows by Sequential S t e p s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Angiogenesis Is a Preneoplastic Marker. . . . . . . . . . . . . . . . . . . . . . . . . Solid Tumors Are Angiogenesis Dependent . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . The Vascularized Tumor Continues to Alter Its Blood Supply.. . . . . . . . . . . . . . . . Mast Cells and Heparin Can Potentiate Tumor Angiogenesis Angiogenesis Can Also Be Induced by Certain Nonmalignant Angiogenic Factors and Endothelial Mitogens Have Been Isolated from Tumors and from Some Nonneoplastic Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , . , , . A. Tumor-Derived Angiogenic Factors B. Angiogenic Factors Derived from Nonneoplastic Cells and Tissues. . . . . . . . . Angiogenesis Inhibitors Are Found in Natural Sources Role of Angiogenesis in Clinical Oncology.. . . . . . . . . . Summary References. . . . . . , . , . . , . . . . . . . . . . . . , . . , . , . . . . . . , . . . . , . . . . . . . . . , . , , . , . , , ,

175 176 176 177 177 178 178 180 181 183 184 185 187 188 189 191 196 198 199

I. Introduction

Angiogenesis is the process of generating new capillary blood vessels and leads, therefore, to neovascularization. Angiogenesis occurs during embryonic development (Wagner, 1980; Bar, 1980) and during several physiological and pathological conditions in adult life. For example, ovulation and wound healing could not take place without angiogenesis (Jakob et al., 1977; Gospodarowicz and Thakral, 1978; Hunt et al., 1981). Angiogenesis is also associated with chronic inflammation (Fromer and Klintworth, 1975) and with certain immune reactions (Sidky and Auerbach, 1975). Many nonmalignant diseases of unknown cause are dominated by angiogenesis. For example, the neovascularization associated with retrolental fibroplasia or with diabetic retinopathy may lead to blindness in both cases. New capillaries may invade the joints in arthritis (Folkman et al., 1980). Solid tumors induce angiogenesis. However, tumor angiogenesis differs at 175 ADVANCES IN CANCER RESEARCH, VOL 43

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least in a temporal way from the other types of angiogenesis described. In physiological situations, as for example in the development of the corpus luteum or in ovulation, angiogenesis subsides, or is turned off, once the process is completed. In certain nonmalignant processes, angiogenesis is abnormally prolonged, although still self-limited, as in pyogenic granuloma or keloid formation. By contrast, tumor angiogenesis is not self-limited. Once tumor-induced angiogenesis begins, it continues indefinitely until all of the tumor is eradicated or until the host dies. Recent progress in understanding the role of angiogenesis in progressive tumor growth will be discussed in this article. It is important to recognize that the phenomenon of angiogenesis is less accessible to investigation than, for example, blood coagulation. Progress in this field was hampered until new methods were developed for the study of capillary growth in vivo and in vitro. II. Methods for Studying Angiogenesis

The early literature on tumor angiogenesis is mainly descriptive, and most investigators took advantage of transparent chambers that could be implanted in animals. The first such chamber, developed by Sandison in 1928 (Sandison, 1928), was implanted in the ear of a rabbit. New vessels grew into the chamber in response to the wound of implantation. Later, tumors were inserted into the chambers. (For an excellent review see Peterson, 1979.) For example, Algire et uZ. (1945) concluded from chambers implanted in mice that tumors could continuously induce the growth of new blood vessels. In 1968, Greenblatt and Shubik (Greenblatt and Shubik, 1968) used a similar chamber in the hamster cheek pouch to demonstrate that capillary proliferation was still induced by a tumor even when it was separated from the host’s vascular bed by a millipore filter. In the 1970s, new techniques were needed to quantitate capillary growth and to test multiple fractions of tumor extracts for angiogenic activity. Four methods developed by Folkman and his associates are now employed by many investigators who study angiogenesis. These are described below. A. THE CORNEAL MICROPOCKET The corneal micropocket technique permits the linear measurement of individual growing capillaries. Tumor implants (1 mm3) are inserted into a pocket made in the cornea of a rabbit at a distance of 1 to 2 mm from the edge of the cornea and the normal vascular bed (Gimbrone et al., 1974). New capillaries grow at right angles from the edge of the cornea and elongate toward the tumor at approximately 0.2 mm/day. They are measured with a

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slit-lamp stereoscope. Other tissues can be interposed between the tumor and its vascular bed. For example, the angiogenesis inhibitory property of cartilage was demonstrated in this way (Brem and Folkman, 1975). A disadvantage is that tumors other than those of rabbit origin may excite an immune response once they are vascularized. Subsequent immunological neovascularization may confuse the original experiment. This problem has been overcome at least for mouse tumors by the implantation of tissues into the cornea of inbred mice (Muthukkaruppan and Auerbach, 1979). The rat cornea has also been used (Fournier et al., 1981). Another pitfall is nonspecific inflammation. Inflammatory agents usually can be defined by their ability to attract neutrophils, lymphocytes, or macrophages into the cornea. These cells may themselves elicit neovascularization. For example, solutions that are hyperosmotic or of abnormally low or high pH are inflammatory. Whenever a new substance is tested for angiogenic activity, it is important to obtain histological sections to determine if an inflammatory response is present.

POLYMERIMPLANTS B. SUSTAINED-RELEASE After the corneal micropocket technique was developed, it became feasible to substitute soluble tumor extracts for tumor implants. However, most of these extracts rapidly diffused away. The problem was how to release these extracts over a sustained period of time so that a concentration gradient could be established within the cornea. There were two further requirements: (1)the implantable sustained-release vehicle had to be inert and could not itself cause inflammation in the cornea, and (2) the implant had to be capable of releasing substances of large molecular weights. After empirical testing of a variety of polymers, two were found to satisfy these conditions: poly(2-hydroxyethyl methacrylate) and ethylene-vinyl acetate copolymer (Langer and Folkman, 1976). From such implants it has been possible to release proteins and other macromolecules at nearly constant rates at micrograms/day and, more recently, nanogramstday (Murray et al., 1983a)for periods of weeks to months. Implants can be as small as 1mm3 and are well tolerated in the cornea and other tissues.

C. CHICKEMBRYO CHORIOALLANTOIC MEMBRANE As biochemists attempted to purify angiogenic activity from extracts of tumor and normal tissue, a more rapid assay was needed to screen numerous fractions. A method of dropping the chorioallantoic membrane and opening the egg shell to reveal a large expanse of vascular membrane was previously described by Leighton (Leighton, 1967). The vessels of the chorioallantoic

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membrane grow rapidly until day 11, after which endothelial proliferation rapidly decreases (Ausprunk and Folkman, 1977). Tumors or tumor fractions implanted on day 9 induce an angiogenic response within 48-72 hr. This response can be recognized under a stereoscope as new capillary loops converging on the implant (Knighton et al., 1977; Klagsbrun et al., 1976). Two vehicles are commonly used for the delivery of small quantities (1-50 pg) of test material over these relatively brief periods of time. The test substance can be dissolved in 0.5% methyl cellulose, which is then dried to make disks of about 2 mm diameter (Taylor and Folkman, 1982). An alternative method is to dry the test substance in 5-10 pl of distilled water on a Thermanox plastic coverslip. Either disk is then applied to the chorioallantoic membrane. Recently it was found that the addition of 1-2 units of heparin (6-12 Fg) to the test substance potentiated the angiogenesis so that the assay could be read in 1 or 2 days (Taylor and Folkman, 1982; Folkman et al., 1983). Another recent finding is that angiogenesis inhibitors can easily be tested on the 4-day yolk sac vessels or on the 6-day chorioallantoic membrane vessels of shell-less embryos cultured in petri dishes (Taylor and Folkman, 1982; Folkman et al., 1983).

D. CLONEDCAPILLARY ENDOTHELIAL CELLS Capillary endothelial cells have been cloned and passaged in long-term culture (Folkman et al., 1979). These cells are useful for detecting endothelial cell mitogens. They form tubes and branches in uitro (Folkman and Haudenschild, 1980; Madri and Williams, 1983) and further buttress in vivo observations that suggest that capillary growth is a multistep process requiring an orderly sequence of events. Tube formation in uitro has also been observed with endothelial cells from umbilical vein and fetal aorta (Maciag et al., 1982; Feder et al., 1983).

Ill. A Capillary Grows by Sequential Steps

By using all of these techniques to study angiogenesis, it is becoming clear that capillary growth takes place by a series of sequential steps that are similar regardless of' the type of angiogenic stimulus. These steps can be summarized as follows: 1. New capillaries originate from small venules or from other capillaries. Larger vessels with layers of smooth muscle do not usually give rise to capillary sprouts. 2. Local degradation of the basement membrane on the side of the venule closest to the angiogenic stimulus (Ausprunk and Folkman, 1977) is one of the earliest events. Capillary endothelial cells stimulated in uitro by an-

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giogenic substances secrete high concentrations of collagenase and plasminogen activator (Moscatelli et al., 1981; Gross et aZ., 1983). These findings suggest that the local degradation of basement membrane seen in viuo is carried out directly by endothelial cells once they receive an angiogenic stimulus. Additional evidence for the availability of stimulated endothelial cells to degrade basement membrane is reported by Madri and Williams (1983) and by Kalebic et al. (1983). 3. Through this opening in the basement membrane, endothelial cells begin to migrate toward the angiogenic stimulus (Ausprunk and Folkman, 1977). 4. Endothelial cells that follow the leaders align in a bipolar fashion as the first sprout begins to form. This alignment has also been demonstrated in uitro, in capillary sprouts growing in plasma clots (Nicosia et al., 1983). 5 . Lumen formation begins. In most examples of postembryonic angiogenesis, a lumen appears to be produced by a curvature that develops in the capillary endothelial cell as if the cytoskeleton itself were undergoing curvature. This phenomenon has been observed in viuo in the cornea (Ausprunk and Folkman, 1977) and also in uitro (Folkman and Haudenschild, 1980). However, during early embryonic angiogenesis, lumen formation may take place by vacuole formation. This has also been observed in viuo (Bar and Wolff, 1972) and in uitro (Folkman and Haudenschild, 1980). 6. Endothelial cells in the midsection of the sprout begin to undergo mitosis. The leading capillary endothelial cells at the very tip of the sprout continue to migrate, but usually do not divide. 7. Loop formation occurs next, as individual sprouts join or anastomose with each other. These loops then elongate and may be the origin of additional sprouts. How individual sprouts find each other is not known. The loops continue to converge upon the angiogenic target. 8. Flow begins slowly after loops have formed. 9. Pericytes emerge along the length of the capillary sprout. 10. Synthesis of new basement membrane follows. When cloned capillary endothelial cells were studied in uitro, most of these same events were observed. Entire capillary networks developed in culture dishes and included lumina, branches, and multiple layers (Folkman and Haudenschild, 1980; Madri and Williams, 1983; Montesano et al., 1983). Maciag, using umbilical vein endothelial cells, has shown that these in uitro “tubes” are hollow and will carry fluid (Maciag et al., 1982). Pericytes were not present in uitro. When the observations from these studies are taken together with in uiuo observations, they suggest that the vascular endothelial cell expresses a defined program of events to generate a capillary network. The program seems to be the same regardless of whether the angiogenic signal is from a tumor or from an inflammatory agent or an immunological stimulus.

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IV. Angiogenesis Is a Preneoplastic Marker

In a series of carefully designed experiments, Gullino and his associates demonstrated that angiogenic activity is acquired (or markedly increased) during progression of normal cells to the neoplastic state (Gimbrone and Gullino, 1976; Brem et al., 1977; Maiorana and Gullino, 1978). In mice and rats, the resting adult mammary gland has practically no angiogenic activity when tested in the anterior chamber of the rabbit eye. However, mammary carcinomas acquire the capacity almost consistently. Certain hyperplastic lesions develop in the mammary gland of both species (DeOme et al., 1959). These lesions can be transplanted into the mammary fat pad and grow in it to a limited extent. For some of these transplants it is possible to predict quite accurately the frequency of neoplastic transformation (Medina, 1973). The lesions with a high frequency of neoplastic transformation induced angiogenesis at a much greater rate than did lesions of low frequency of transformation. This elevated angiogenic capacity was observed long before any morphologic sign of neoplastic transformation was apparent (Gimbrone and Gullino, 1976; Maiorana and Gullino, 1978). In fact, hyperplastic lesions of the human breast behaved similarly, showing the appearance of strong angiogenic activity long before the onset of malignancy (Brem et al., 1978). Preneoplastic lesions of human bladder mucosa also display high angiogenic activity in contrast to benign lesions that have little or no angiogenic activity (Chodak et al., 1980). Two recent reports provide additional evidence that expression of angiogenic activity (or amplification of angiogenic activity) may appear in cells long before they reach the stage of tumor formation. In one such experiment, the appearance of angiogenesis predicted the later formation of sarcomas around plastic implants in rodents (Ziche and Gullino, 1981). In a second study, normal mouse diploid fibroblasts were carried in culture (see Fig. 1).At each passage, i.e., approximately once per week, the cells were tested for angiogenic activity in the rabbit eye and for tumorgenicity by reimplantation into the mouse strain that donated the fibroblasts (Ziche and Gullino, 1982). Angiogenic activity first appeared at the fifth passage in some dishes and was present in virtually all cells by the seventh passage. Angiogenic activity persisted in all cells thereafter. However, tuinorigenicity did not occur until the fifteenth passage. It should be recognized that angiogenic capacity and neoplastic transformation are probably not interdependent events. Each can be expressed in the absence of the other (Brem et al., 1978). Certain benign tumors can stimulate intense angiogenesis, but do not become malignant, for example, adrenal adenomas. Furthermore, “tumor take” does not require angiogenesis.

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PASSAGE NUMBER

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FIG. 1. Summary of experiment by Ziche and Gullino (1982) demonstrating that angiogenic capacity precedes tumorigenicity. Normal mouse diploid fibroblasts are harvested from inbred mice and cultured. At each weekly passage, the cells are reimplanted into the same mouse strain to test for turnorigenicity. The cells are also implanted into the rabbit cornea to test for angiogenic capacity. (The rabbits were pretreated with corticosteroids to prevent inflammatory or immune angiogenesis.) By 5 weeks, cells in some of the culture plates are angiogenic hut none is tumorigenic. By the seventh passage, virtually all of the cells are angiogenic. However, tumorigenicity does not appear until the fifteenth week.

V. Solid Tumors Are Angiogenesis Dependent

A clue that tumor growth might be dependent upon neovascularization came from observing the growth of tumors implanted into organs maintained by isolated perfusion in glass chambers (Folkman et al., 1963, 1966; Folkman, 1970). New capillaries could not proliferate from the vascular network ofthese isolated organs. This was due to gradual endothelial degeneration, a common problem of isolated, perfused organs (Gimbrone et al., 1969). The tumor implants remained “avascular” and stopped growing at diameters less than 23 mm. Nevertheless, when these avascular tumors were transplanted back into mice, they grew, became vascularized, and eventually killed their host. From these experiments taken together with those of Algire et al. (1945),a hypothesis was proposed that tumors are angiogenesis dependent (Folkman, 1972). In its simplest terms, this hypothesis can be stated: Once tumor take has occurred, every increase in tumor cell population must be preceded by an increase in new capillaries that converge upon the tumor. The hypothesis also implies that angiogenesis is a control point common to most, if not all, solid neoplasms. Evidence in support of this idea was accumulated from four types of experiments:

1. The prevascular phase can be measured and prolonged in uivo. A discrete avascular phase of tumor growth exists during which tumors are usually microscopic or at least not palpable. In the conventional transplanta-

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tion of tumor into experimental animals, this phase is brief (3 to 6 days) and generally indiscernible. However, the avascular phase can be studied comfortably when the tumor is separated from its vascular bed. The severe limitation of tumor growth in the absence of angiogenesis is then immediately appreciated. For example, tumors implanted into the anterior chamber of the rabbit eye will float in the aqueous humor and reach mean volumes of approximately 0.5-0.6 mm3 by 14 days. In contrast, the same tumors subsequently seated on the iris become vascularized rapidly and attain a mean volume of approximately 330 mm" during the same period of time (Gimbrone

et al., 1972). 2. The prevascular phase can be simulated by an in vitro model. Multicellular tumor spheroids suspended in soft agar (Sutherland et al., 1971) can

be used as an approximate model of the prevascular phase of tumor growth by changing the medium frequently (Folkman and Hochberg, 1973; Folkman et aZ., 1974). Spheroids from a variety of different tumor types at first enlarge exponentially, then gradually slow their growth, and reach a critical diameter beyond which there is no further enlargement. For example, B16 melanoma reaches a mean diameter of approximately 2.4 min. At this steady state, viable cells proliferate in the periphery while cells in the center undergo necrosis. The mechanism of this dormancy appears to be a combination of limited inward diffusion of oxygen (once the spheroid's radius exceeds approximately 150-200 pm) and limited outward diffusion of metabolic wastes. (For a detailed study of the mechanism of growth arrest in these spheroids see Carlsson et d.,1979.) 3. The effects of the vascular phase upon tumor growth can be observed. Once the vascular phase has commenced, tumor cells lying closest to an open capillary have the highest [3H]thymidine-labeling index. The labeling index in tumor cells decreases as they increase their distance from an open capillary (Tannock, 1968). This relationship of tumor cell proliferation to proximity of capillaries within the tumor correlates well with the estimated oxygen diffusion lengths from the center of a given capillary. Thus, it appears that solid tumors are made up of cylinders or cords of tumor cells surrounding individual capillary units. Denekamp (1982) has shown that this relationship also holds for human tumors. That tumor cells prefer contiguity to capillaries may not wholly depend on diffusion of oxygen and nutrients. Nicosia et al. (1983) implanted small fragments of rat aorta into plasma clots, and capillaries grew out radially. When tumor cells were inoculated into the periphery of the plasma clot, they grew slowly as a small spheroidal focus. However, when a capillary sprout approached the tumor focus, tumor cells grew rapidly around the capillary as a cylindrical cuff. Tumor growth extended toward the center of the plasma clot (where the aortic segment had been placed). There is no blood flow in this system. One interpretation is

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that tumor growth can be directly facilitated by capillary endothelial cells or perhaps by the matrix which they produce. 4. The administration of angiogenesis inhibitors stops tumor growth. Certain angiogenesis inhibitors now available can be administered locally, regionally, or systemically and can prevent tumors from becoming vascularized. Thus, some tumors can now be maintained in the prevascular phase or eradicated by angiogenesis inhibition alone. These experiments will be discussed in Section X (see also Langer et al., 1980; Taylor and Folkman, 1982; Folkman et al., 1983). In summary, it should be remembered that the dependency of solid tumors on angiogenesis is related to the growth of their cells in a tightly packed population of high density (10R-109 cells/cm3)).Wherever malignant cells have developed the capacity to grow separately from each other (for example, as ascites or as infilitrates in certain leukemias), angiogenesis is not required. (There are, of course, many ascites tumors that retain the capacity to induce angiogenesis, and these cells can grow in both the solid and ascites configurations. Examples are Walker carcinoma in rats and ovarian carcinoma in humans.) VI. The Vascularized Tumor Continues to Alter Its Blood Supply

At first glance, a growing tumor whose cells are stimulating new capillary sprouts and then accumulating around them appears analogous to an organ in the embryo. But the analogy does not hold because tumors continue to alter their intrinsic vasculature in a way that normal organs do not. For example, tumors tend to increase their vascular volume. Thus, in rats where vascular volume is 20%of normal tissue weight, it is 50% of tumor weight in the early stage of hepatoma (Yamaura and Sato, 1974). While the increase in vascular volume is different from one tumor type to another, specific tumor types tend to maintain a characteristic increase in vascular space (Gullino and Grantham, 1964). Second, tumors eventually begin to compress their own capillaries. In most experimental tumors of a size less than 0.5 cm3, new vessels are open throughout the tumor, and there is no necrosis. Beyond this size, there may be gradual compression of capillaries. Because this extravascular pressure can close capillaries and stop the microcirculation (Warren, 1970), it is more accurate to envisage that tumors compress their blood supply rather than to perpetuate the notion that tumors outgrow their blood supply. This compression eventually leads to prolonged cessation of flow in the core of the tumor followed by central necrosis. Central necrosis begins to appear for a variety of experimental tumors after

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they have grown beyond approximately 1-2 om3 Goldacre and Sylven, 1962). What accounts for this compressive effect? Mitosis contributes. After each cell division, the added pressure from daughter cells must be attenuated throughout the tumor mass. Leaky capillaries also contribute. New capillaries in the tumor leak fluorescein and colloidal carbon which normal capillaries do not. This increased permeability of new capillaries is compounded by the lack of lymphatics in most tumors (Swabb et al., 1974). We have never found lymphatics in tumor implants in the rabbit cornea, even after these implants have become heavily vascularized. As a result, extravascular tissue pressure is higher in a tumor than in its normal counterpart (Young et al., 1950; Peters et al., 1980; Paskins-Hulburt et al., 1982). The increased leakiness of capillaries within a tumor bed may be facilitated by a tumor factor that increases permeability (Dvorak et al., 1981). Also, in certain tumors (e.g., brain tumors), structural alterations appear in the new capillaries. Fenestrations arise in the endothelial cells, pinocytotic vesicles are increased, and occasionally there are interruptions in the basement membrane (Hirano and Matsui, 1975; Pousa et al., 1979; Deane and Lantos, 1981). VII. Mast Cells and Heparin Can Potentiate Tumor Angiogenesis

There is increasing evidence that the mast cell may act as a helper to the capillary endothelial cell during certain types of angiogenesis, especially tumor angiogenesis. In his first description of mast cells, Ehrlich (1879) noted that they seemed to be more frequent in the neighborhood of carcinomas. While increased mast cell populations are found in a variety of pathological conditions including immediate hypersensitivity or chronic inflammation, they have also been shown to congregate in areas of neovascularization. Increased mast cells have been reported in the most highly vascularized areas of certain tumors (Giani, 1964), at the periphery of carcinoma in situ (Dunn and Montgomery, 1957), and in the axillary nodes of patients with breast cancer metastatic to the nodes (Thoresen et al., 1982). These correlations led to the notion that mast cells might somehow be associated with the growth of new capillaries, although just what this association might be was not known (Ryan, 1970; Smith and Basu, 1970). In an attempt to quantitate the relationship of mast cells to neovascularization, tumors were implanted on the chorioallantoic membrane of the chick embryo (Kessler et al., 1976). There was a 40-fold increase in mast cell density in the area of the tumor implant. These mast cells appeared in the neighborhood of the tumor about 24 hr before new capillary sprouts. While mast cells alone were unable to induce angiogenesis, cultured mast cells or the media in which they incubated stimulated migration of capillary endo-

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thelial cells in uitro (Azizkhan et al., 1980). Heparin released by the mast cells was responsible for this stimulation of capillary endothelium (Azizkhan et al., 1980). Furthermore, heparin potentiated tumor angiogenesis. When the chorioallantoic membrane is exposed to tumor extracts with angiogenic activity, neovascularization is usually observed in about 3 days. However, if a small amount (6-12 pg) of heparin is added to the tumor extract, angiogenesis is potentiated and neovascularization appears within 1day (Taylor and Folkman, 1982; Folkman et al., 1983). Heparin by itself does not induce angiogenesis. However, heparin does become angiogenic when it is complexed to copper (Ziche et al., 1981). The mechanism of this phenomenon is unknown. These findings will be discussed in more detail in Section IX. VIII. Angiogenesis Can Also Be Induced by Certain Nonmalignant Cells

Several types of normal cells are known to induce angiogenesis under appropriate conditions. For example, macrophages, when properly activated, can release angiogenic activity in the cornea (Polverini et al., 1977). Macrophages are activated in uiuo by injecting paraffin oil or endotoxin into the peritoneal cavity of experimental animals or in uitro by adding latex beads to the culture medium. Macrophages collected from wounds display similar angiogenic activity (Thakral et al., 1979; Hunt et al., 1981). Recently it has been shown that wound macrophages are oxygen sensitive in relation to angiogenic activity. When these macrophages are activated by wound debris or fibrin products, they produce maximal angiogenic activity if oxygen concentration is minimal. As local oxygen tension rises (for example, after new capillaries appear in the wound), macrophage angiogenic activity is decreased and eventually turned off (Banda et al., 1982; Knighton et al., 1983). Macrophages are attracted to some, but not all, tumors (Mantovani, 1982). Polverini and Leibovich (1984) have recently reported that macrophages embedded in a tumor may also contribute to its angiogenic activity. The idea that other types of'leukocytes might also have angiogenic activity arose from the observation of corneal neovascularization associated with leukocyte infiltrates (Fromer and Klintworth, 1975). Lymphocyte-induced angiogenesis was observed when allogeneic immunocompetent lymphocytes were injected intradermally into immunosuppressed or irradiated host mice. The phenomenon has been most clearly defined by Auerbach and his associates and by subsequent workers (Sidky and Auerbach, 1975; Fromer and Klintworth, 1975; Kaminski et al., 1975; Auerbach, 1981). Not all classes of lymphocytes are able to induce angiogenesis, however. For example, angiogenesis is evoked by effector T cells (i.e., corticosteroid-resistantthymocytes or by spleen or lymph node cells), but not by spleen cells from

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athymic (nulnu)donor animals (Sidky and Auerbach, 1975; Kaminiski et aZ., 1978a). The most dramatic lymphocyte-induced angiogenesis is achieved by donor lymphocytes that differ at the H - 2 locus from the recipient animal (Auerbach and Sidky, 1979). Taken together, these results indicate that the cell responsible for lymphocyte-induced angiogenesis belongs in the general class that includes most of the lymphokine-producing T cells (Adelman et al., 1980). However, the mechanism of lymphocyte-induced angiogenesis is not yet clear. It is not known whether foreign lymphocytes can stimulate endothelial cells directly or whether lymphocyte angiogenic activity is mediated through another cell such as an activated macrophage. Fat cells or adipocytes derived from 3T3 fibroblasts that have undergone differentiation in uitro (Green and Kehinde, 1976) are also capable of inducing angiogenesis (Castellot et al., 1980). Angiogenic activity is differentiation-dependent because adipocytes secrete more angiogenic activity than preadipocytes. These 3T3 adipocytes secrete in a differentiation-dependent fashion a factor that stimulates chemotaxis of vascular endothelial cells in uitro and neovascularization in uiuo (Castellot et aZ., 1982). Conditioned medium from adipocytes, but not from preadipocytes, strongly stimulates both plasminogen activator and collagenase release from capillary endothelial cells, but not from aortic endothelial cells (Rifkin et al., 1982). Taken together, these findings provide the first demonstration of an angiogenic activity secreted as a consequence of the dqferentiation of the producer cell type. At least six normal tissues so far have been demonstrated to express angiogenic activity during some period of their adult life. For some tissues, this angiogenic activity is expressed briefly or in a cyclic manner. For each of the tissues, the cells responsible for angiogenesis are as yet unknown. For example, during the period when a follicle forms in the ovary and the corpus luteum develops, the corpus luteurn is quickly invaded by new capillary sprouts. At this stage, the corpus luteum can express angiogenic activity when transplanted, for example, to the hamster cheek pouch or to the rabbit cornea (Jakob et al., 1977; Gospodarowicz and Thakral, 1978). Human follicular fluid is also angiogenic (Frederick et d.,1984). When the corpus luteum regresses, the new vessels also regress. Testicular fragments from 1day-old mice implanted into the subcutaneous tissue of castrated mice of the same strain can also induce transient angiogenesis (Huseby et al., 1975). Fragments of kidney from newborn hamsters produce a weak angiogenesis response in the hamster cheek pouch even when enclosed by a millipore filter (Warren et al., 1972). Fragments of embryonic or adult mouse kidney also give a weak angiogenic response on the chorioallantoic membrane of the chick embryo (Folkman and Cotran, 1976).

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Under certain conditions epidermis, but not dermis, is angiogenic (Nishioka and Ryan, 1972; Wolf and Harrison, 1973). Fragments of retina placed into a corneal pocket in rabbits also induce neovascularization, whereas other ocular tissues such as sclera have no activity or are weakly angiogenic (G. C. Brown, 1980). Glaser et al. (1980a) isolated a vasoproliferative factor from mammalian retina. Further purification of this activity from bovine retina has been reported and will be discussed in the following section on angiogenic factors (D’Amore et al., 1981). Extracts of the male mouse salivary gland were found to induce angiogencsis in the chick embryo (Folkman and Cotran, 1976). However, this observation has not been pursued because of the inflammatory reaction associated with it. IX. Angiogenic Factors and Endothelial Mitogens Have Been Isolated from Tumors and from Some Nonneoplastic Cells

In the previous discussion we have seen that a better understanding of the phenomenon of angiogenesis has been achieved by the study of its component events. It is more difficult to describe angiogenesis in terms of its chemical mediators. However, progress is gradually being made from the contributions of many laboratories. In some respects, the attempt to understand the chemical mediators of angiogenesis is analogous to the current effort to understand classic immunological phenomena in terms of the chemical factors that mediate them. However, the elucidation of angiogenesis factors is formidable because the bioassay for angiogenesis is carried out only in uiuo. Furthermore, even if neovascularization results from the application of a test substance to such an in uiuo assay, the substance cannot immediately be labeled as an angiogenic factor. It could, by causing injury or inflammation, act as a chemotactic factor for other cells such as macrophages, which themselves might then be the source of angiogenic activity. Formic acid is an example. To avoid this confusion the concept of direct and indirect angiogenic activity was introduced (Folkman and Haudenschild, 1980). A “direct” angiogenic factor could stimulate capillary proliferation without having to depend on intervening cell types. Careful histological monitoring, at present, is about the only way of distinguishing between direct and indirect angiogenesis. Another difficulty is that components of angiogenesis, such as endothelial proliferation, migration, or enzyme production, can be studied individually in uitro, but they do not necessarily predict angiogenic activity in uiuo. Thus, a factor that stimulates endothelial mitosis in uitro may or may not also be angiogenic.

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With these caveats in mind, the following discussion summarizes angiogenic factors that have been reported to date. A. TUMOR-DERIVED ANCIOGENICFACTORS

The first angiogenic factor was isolated from Walker 256 carcinoma grown in rats (Folkman et al., 1971). The angiogenic activity was isolated from cells in the ascites form, and this activity was destroyed by proteases or by heating. In a subsequent report, similar activity was isolated from the nuclei of these tumor cells and was associated with the nonhistone proteins (Tuan et al., 1973). Phillips et ul. (1976) extracted angiogenic activity from solid Walker tumor cells and found two high-molecular-weight fractions, one of which (35,000 to 100,000) gave the most intense angiogenic activity. Rat liver or regenerating liver handled in the same way was not active. Angiogenic activity was also found in a human Wilms’ tumor and in a hypernephroma. Fenselau and Mello (1976; Fenselau et al., 1981a) also used Walker tumor cells, but guided purification by in vitro assays based on proliferation of fetal bovine aortic endothelial cells. Periodically the fractions were tested for angiogenic activity in the chick embryo and in the rat cornea. In their early reports, angiogenic activity from homogenates of ascites tumor cells appeared in fractions of high molecular weight when analyzed by gel filtration and neutral pH. However, at p H 4, angiogenic activity was associated with materials of molecular weight less than 800. Further purification of these materials by silica gel chromatography of ethanol extracts of the lyophilized tumor homogenates revealed a low-molecular-weight material with an ultraviolet absorption maximum near 260 nm which was not composed of protein or peptides (Watt, 1981; Fenselau, et al., 1981b; Fenselau, 1984). A factor isolated from the media of cultured neural cells and neural tumor cells stimulated proliferation of umbilical vein endothelial cells in culture (Suddith et ul., 1975). In a similar experiment, an endothelial mitogen for aortic endothelial cells was isolated from cultures of Walker tumor cells (McAuslan and Hoffman, 1979). In another study, a factor isolated from Walker tumor cells was mitogenic for microvascular endothelial cells derived from cow brain and was also angiogenic in the chick embryo (Schor et al., 1980).This factor and the one reported by McAuslan were both of low molecular weight. Angiogenic activity was found in the cultured supernate of seven cell lines derived from a variety of spontaneous human tumors and grown in largescale suspension culture. Activity was not further purified (Tolbert et al., 1981). The low-molecular-weight angiogenic factor (approximately 200) pre-

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viously found in Walker tumor cells by Schor et aZ. (1980) stimulated capillary endothelial cells but not aortic endothelial cells, and then only when the capillary cells were growing on native collagen substratum (Keegan et aZ., 1982). Most recently, this group in Kumar’s laboratory has grown a human lung tumor in serum-free medium for 12 months and has isolated an angiogenic factor from the medium (Kumar et al., 1983a). The highest angiogenic activity was in the range of approximately 80,000 MW. Also, a dialyzable, low-molecular-weight angiogenic factor was found in this medium. These authors concluded that the high-molecular-weight fraction possibly contained a carrier protein and the low-molecular-weight fraction was carrier-free. The low-molecular-weight component did not absorb at 260 nm and was felt to be different from that isolated by Fenselau et al. (1981b). This study also ruled out the possibility that angiogenic activity could be a component of serum because the cells were grown in serum-free media. Angiogenic activity has also been isolated from human central nervous system tumors in culture (Matsuno, 1981) and from cultures of human malignant melanoma cells (Stenzinger et aZ., 1983). However, neither of these angiogenic activities has been purified. Recently, a tumor-derived endothelial mitogen that is angiogenic has been purified to homogeneity (Shing et al., 1983, 1984). This factor was obtained from chondrosarcoma grown in the rat. The factor was found in extracts of the tumor cell but also from extracts of the chondrosarcoma matrix. It was purified one million-fold to a single-band preparation by a two-step procedure that utilized cationic exchange on Biorex 70 and affinity chromatography by heparin-Sepharose. The purified factor is a cationic peptide with an isolectric point of approximately 9.8 and a molecular weight of about 18,000 (Fig. 2). It stimulates capillary endothelial proliferation halfmaximally at a concentration of 1ng/ml. It stimulates strong angiogenesis on the chorioallantoic membrane of the 9-day-old chick embryo at concentrations of 120 ng within 24 hr. Histologic sections reveal that neovascularization takes place in the virtual absence of inflammatory cells. It is too early to say which of these tumor-derived endothelial mitogens and angiogenic factors are, in fact, responsible for the angiogenesis induced by growing tumors. It is also premature to predict which factors may be structurally similar to each other. B. ANGIOGENIC FACTORS DERIVEDFROM NONNEOPLASTIC CELLS AND TISSUES Angiogenic activity has been isolated and partially purified from synovial fluid (R. A. Brown et al., 1980),wound fluid (Banda et aZ., 1982), and largescale cultures of granulocytes and monocytes (for review see Wissler, 1982).

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FIG.2. A purified endothelial mitogen derived from rat chondrosarcoma. Chondrosarcoma extracellular matrix was digested with collagenase and purified by Bio-Rex 70 chromatography followed by heparin-Sepharose chromatography as described by Shing et al. (1984).On the left, SDS-polyacrylamide gel electrophoresis. (Slot 1) Molecular weight markers (BRL). (Slot 2) The peak fraction of growth activity (200 ng = lo00 U). This purified growth factor was added at various concentrations to cultures of bovine capillary endothelial (BCE) cells and proliferation measured. Half-maximal stimulation is induced by growth factor concentrations of about 1 ng/ml. About 600 U activity (120 ng) are required to produce strong angiogenesis within 24 hr on the 9-day-old chick chorioallantoic membrane. Histological sections reveal that inflammation is virtually absent. The recovery of activity from the tumor is about 5% and the yield of pure growth factor is about 1 pg from 5 g of crude chondrosarcoma matrix.

The mammalian retina has also been a rich source of angiogenic activity, the first report of which was by Glaser et al. (1980a,b). In subsequent reports, this retinal angiogenic factor has been further characterized and further purified (D'Amore et al., 1981). These factors obtained from bovine retina were of large molecular weight. However, a low-molecular-weight angiogenic factor was isolated from cat retina (Kissun et al., 1982). Also, an angiogenic factor in bovine retina was found to possess at least one common antigenic determinant with an angiogenic factor from a tumor extract (Shahabuddin and Kumar, 1983). A factor has been derived from the medium of cultured 3T3 adipocytes that stimulates neovascularization as well as chemotaxis of capillary endothelial cells (Castellot et al., 1982). Angiogenic activity has also been extracted recently from human myocardial infarcts taken postmortem (Kumar et al., 1983b). Partial purification demonstrated a low-molecular-weight factor of approximately 300, analogous to the molecular weight of tumor an-

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giogenesis activity obtained from Walker tumor cells by this group of investigators. Prostaglandin E, (PGE,) has also been correlated with angiogenic activity in the cornea (BenEzra, 1978; Ziche et al., 1982), and prostaglandin E, (PGE,) has been shown to have angiogenic activity on the chorioallantoic membrane (Form and Auerbach, 1983). There is also reported to be a relationship between copper ions and angiogenesis. McAuslan (1980) first suggested that copper might play a role in mediating angiogenesis. Subsequently, Raju et al. (1982) showed that either heparin or the tripeptide glycylhistidyllysine, when bound to copper became angiogenic. Copper-free molecules were not angiogenic. At this writing, only one angiogenic factor has been purified to homogeneity. Much work remains to be done (1) to purify each of the reported angiogenic factors; (2) to determine which factors act directly on endothelial cells (or pericytes) and which act as chemotactic agents for other cells (such as mast cells); and (3) to ascertain which factors might actually be participating in tumor angiogenesis or in the angiogenesis of injury. A central question is, if it were possible to neutralize any of these activities (for example, with antibodies), for which factor(s) would such neutralization lead to the inhibition of angiogenesis? Hopefully, in the future it will be possible to sort out the bewildering diversity of factors that influence endothelial and capillary growth. Perhaps we will learn that some angiogenic factors are first synthesized as highmolecular-weight species from which smaller active units are cleaved. X. Angiogenesis Inhibitors Are Found in Natural Sources

The concept of “antiangiogenesis” as a potential therapeutic approach was put forward in 1972 (Folkman, 1972). At the time, however, there was no known angiogenesis inhibitor. Clinical observations suggested that if an angiogenesis inhibitor existed at all, cartilage would be a good place to look for it. For example, osteogenic sarcoma of the bone rarely spreads to adjacent cartilage; breast cancer metastatic to the vertebral bones rarely invades adjacent cartilage in the vertebral disc. ‘The first experimental evidence for this notion was the demonstration by Eisenstein et al. (1973) that cartilage extracted with guanidine lost its resistance to vascular invasion. Subsequently, Brem and Folkman (1975) showed that an implant of cartilage placed adjacent to a tumor in the rabbit cornea inhibited growth of new blood vessels toward the tumor. A factor was isolated and partially purified from cartilage that when locally administered by a sustained-release polymer, inhibited tumor angiogenesis in the cornea (Langer et al., 1976). This cartilage-derived factor was also infused into the carotid artery of mice and

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rabbits. The factor inhibited gowth of tumor blood vessels and of the tumor itself (Langer et al., 1980). Eisenstein’s group demonstrated that resistance to invasion by both vascular and connective tissue could be diminished if the cartilage were extracted with guanidine hydrochloride (Sorgente et al., 1975). These guanidine extracts contained protease inhibitor activity (Sorgente et al., 1976). The extracts suppressed the growth of fibroblasts and aortic endothelial cells in culture. Kuettner and his associates showed that cartilage also contained inhibitors of collagenase that blocked bone resorption in vitro (Kuettner et al., 1978). The term “antiinvasive factor” (AIF) was used to categorize these inhibitors. Subsequently, Kuettner’s group found that the collagenase secreted into the media by tumor cells was also inhibited by AIF (Kuettner et al., 1977). When human bone explants were cocultured with tumor cells, the tumor cells invaded and eroded the bone but not the cartilage. In fact, tumor cells infiltrated the cartilage matrix only where it had been previously occupied by capillary loops of the growth plate and nutrient canal (Kuettner and Pauli, 1983). These findings were interpreted to mean that vascular endothelial cells and tumor cells may each generate collagenase and that cartilage could resist invasion by both cell types. Recently, Kaminski et al. (1978b) showed that extracts of human cartilage administered intravenously inhibited vasoproliferation in mice. Also, Cawston et al. (1981) and Bunning et al. (1984) have isolated a series of metalloproteinase inhibitors from cartilage that may play an important role in the antiangiogenic properties of cartilage. Murray et al. (1983) and Gabrielides and Rifkin (1983) have reported further on the purification of a collagenase inhibitor from cartilage. The antiinvasion factor of Kuettner et al. is not purified, nor is the angiogenesis inhibitor of Langer et al. It is too early to say whether these two moieties will turn out to be similar; each group uses different bioassays to guide purification. Nevertheless, data from both groups point to cartilage as a major source of angiogenesis inhibitor and suggest that angiogenesis inhibitors may be found in other tissues. For example, Eisenstein et a2. (1979) have reported an angiogenesis inhibitor isolated from the wall of the aorta. This material can inhibit inflammatory angiogenesis in the cornea Medroxyprogesterone acts as an angiogenesis inhibitor when released locally in the cornea in the presence of tumor-induced vessels (Gross et al., 1981). However, when administered systemically, it does not inhibit angiogenesis or tumor growth. Protamine sulfate was the first angiogenesis inhibitor to be effective when administered systemically (Taylor and Folkman, 1982). This discovery arose from a series of experiments on the role of mast cells in angiogenesis which demonstrated the following (see Section VII). (1) Mast cells accumulated at a tumor site before the ingrowth of new capillary sprouts (2) Heparin from

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mast cells increased the migration of capillary endothelial cells in uitro. (3) Tumor angiogenesis was potentiated by heparin in uiuo (4) Protamine blocked the ability of heparin to stimulate migration of capillary endothelial cells. This suggested that protamine or other heparin antagonists might also inhibit angiogenesis in uiuo. Protamine prevented tumor-induced angiogenesis on the chorioallantoic membrane of the chick embryo and it also inhibited growth of embryonic vessels. In the presence of protamine, avascular zones appeared in the 3-day-old yolk sac membranes and in the 6- to 8-day-old chorioallantoic membrane. Protamine had no effect on nongrowing vessels in the chorioallantoic membrane after day 10. Furthermore, protamine-polymer pellets implanted into the rabbit cornea inhibited capillary growth whether induced by tumors, inflammation, or an immunologic reaction (Taylor and Folkman, 1982). When administered systemically to mice, protamine reduced the volume of lung metastases. Many lung tumors remained avascular and stopped growing at mean tumor volumes of 2-3 mm3. Tumor cells were not directly affected by protamine. Tumors implanted in other regions such as the subcutaneous tissue were less responsive to protamine than tumors growing in the lung, probably due to the fact that the uptake of protamine by lung is at least 5 times higher than by subcutaneous tissue and 44 times higher than uptake by skin. It was not possible to raise the dose of protamine sufficiently to inhibit growth of primary tumors or to cause tumor regression because of the toxicity of protamine (lethargy, hypocalcemia, and occasionally sudden death) (Potts et al., 1984). The toxicity is unrelated to antiangiogenesis activity. Despite its toxicity, protamine was the first angiogenesis inhibitor with a known structure. Protamine is an arginine-rich basic protein of 4300 MW found only in sperm. Its amino acid sequence has been determined (Ando and Watanabe, 1969). The experience with protamine suggested that, at best, therapy with angiogenesis inhibitors could reduce a tumor to the avascular stage but could probably not eradicate it. This notion was soon discarded because of the discovery of a more potent form of antiangiogenesis. A new way to inhibit angiogenesis developed as follows. On the basis of the previous finding that heparin potentiated tumor angiogenesis, it was thought that tumor angiogenesis enhanced by heparin might be made more conspicuous in the chick embryo by adding cortisone to suppress background inflammation that occasionally arose as a result of eggshell dust. The result was unexpected. While heparin alone enhanced tumor angiogenesis and cortisone alone had little or no effect, angiogenesis was inhibited by the combination of heparin and cortisone (Folkman et al., 1983). It was further found that heparin administered with cortisone was a potent inhibitor of capillary growth that occurred during embryogenesis (Fig. 3) or that was observed in the cornea from inflammatory or immunological stimuli. Heparin mixed with cortisone in a sustained-release pellet suppressed tumor

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Fic. 3. Inhibition of embryonic angiogenesis. Histologic cross sections (A) of day 8 chorioallantoic membranes. Fertilized chick embryos were removed from their shell on day 3 (or 4) and incubated in a petri dish in high humidity and 3-58 COZ. On day 6 a inethylcellulose disk of approximately 2 mm diameter, previously dried from 10 pl of 0.5% methylcellulose, was implanted on the chorioallantoic membrane. Each disk contained either (1)cortisone acetate, (2) heparin, (3)cortisone + heparin, or (4) cortisone + hexasaccharide. Forty-eight hours later, a clear avascular zone appeared in the membranes exposed to heparin + cortisone but not in the membranes exposed to either compound alone (see below). In the avascular zones, capillaries

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angiogenesis in the rabbit cornea. The anticoagulant function of heparin was not responsible for this antiangiogenic function because a nonanticoagulant fragment, which was a hexasaccharide, substituted for the activity of the parent molecule. The hexasaccharide has a molecular weight of about 1600. Oral administration of heparin to mice and rats resulted in the release of nonanticoagulant heparin fragments in the serum, which in the presence of cortisone administration had similar antiangiogenic effects. Subcutaneous tumors of B16 melanoma, reticulum cell sarcoma, Lewis lung carcinoma, and bladder carcinoma (MB49) regressed in animals drinking heparin and receiving cortisone acetate injections but not in animals receiving heparin or cortisone alone. In fact, it was possible to eradicate tumors completely in more than 50% of mice. The mice remained tumor-free after treatment was stopped. The number of lung metastases in all mice was reduced to 0.1%of the controls. However, in the case of four tumors, neither angiogenesis nor tumor growth was suppressed by optimal combinations of heparin and cortisone. These were sarcoma 1509A, meth A sarcoma, glioma 26, and glioma 261B. It is interesting that these tumors were all induced by the carcinogen 3-methylcholanthrene. Furthermore, when a nonresponding tumor (sarcoma 1509A) was implanted in the left flank of nude mice and a responding tumor such as reticulum cell sarcoma was implanted in the right flank, the reticulum cell sarcoma regressed in each mouse treated with the heparinwere absent, whereas the ectodermal and endodermal layers were present. ~ 5 0 0 (From . Folkman et al., 1983, with permission of the publisher.) Subsequent work has shown that hydrocortisone 21-phosphate (Sigma) provides more reproducible avascular zones than cortisone acetate, presumably because of higher solubility. An optimum concentration is approximately 50 pg. Heparin antiangiogenic activity varies greatly by manufacturer and by batch, and over wide concentration ranges. For example, Abbott Panheprin is optimally effective (with hydrocortisone) at 6-12 pg; Hepar heparin (Franklin, Ohio) is effective from at least 6 to 200 pg with an optimum at approximately 50 pg; Sigma heparin is effective from 6 to 200 pg with an optimum at about 100-150 pg. (B) Avascular zone in 8 day chick embryo chorioallantoic membrane after application of hydrocortisone 21-phosphate (50 kg) and Panheprin (Abbott) (2 units, i.e., approximately 12 pg). X6. After this study was completed in 1983, Panheprin became unavailable because of an earlier decision by the Abbott Company to stop manufacturing heparin. Other beparins such as Hepar, or Sigma, are effective inhibitors of angiogenesis (with corticosteroids) in the chick embryo or the rabbit cornea. However, when administered orally to mice, only Panheprin can bring about the tumor regressions described in the text. The next most potent heparin (Hepar, Inc.), caused regression only of reticulum cell sarcoma, and it and other heparins were generally ineffective against other tumor types. The lack of Panheprin is of no consequence for the demonstration of angiogenesis inhibition in the chick embryo or the rabbit cornea, because a variety of other heparins are effective in these systems. However, until a heparin of antiangiogenic potency equivalent to Panheprin becomes available, or until large quantities of the appropriate hexasaccharide can be easily produced, tumor regression cannot be attained with currently available heparins except in the case of reticulum cell sarcoma.

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cortisone, whereas the sarcoma 1509A continued to grow in the same mouse. Serum from heparin-cortisone or hexasaccharide-cortisone-treated mice was not cytotoxic to tumor cells alone. The inhibitory effect of heparincortisone was specific for growing microvessels; mature, nongrowing vessels remained unaffected. This was demonstrated in the chick embryo at different stages. It is not understood how angiogenesis inhibition results in complete regression of a large tumor mass. However, serial histological studies on a daily basis show progressive loss of capillaries. Residual tumor cells cluster around the remaining capillaries, until finally the entire tumor disappears. If the doses of heparin and cortisone are lowered so that regression is very slow, tumors can be held at a nearly constant size. In some cases, tumors can remain in the avascular state. This implies that with rapid regression, there may be “bystander” killing of residual tumor cells. The mechanism of angiogenesis inhibition by heparin-cortisone or hexasaccharide-cortisone is unknown. However, it has recently been shown (Crum and Folkman, 1984) that neither the glucocorticoid nor the mineralocorticoid activity of cortisone is necessary for antiangiogenesis. For example, a compound such as lla-epicortisol (Upjohn), which has the identical structure to hydrocortisone except that the 11-hydroxyl group is in the a position instead of the f3 position, has no glucocorticoid or mineralocorticoid activity. Yet in the presence of heparin or a hexasacharide fragment of heparin, 11a-epicortisol is a strong angiogenesis inhibitor. The fact that a hexasaccharide fragment of heparin with no previously assigned function, and a corticosteroid with no known biological function can suppress angiogenesis when administered together suggests that regardless of the complexity of the angiogenic phenomenon, it may be governed by simple, naturally occurring molecules. These experimental findings offer a potential fbture role for angiogenesis inhibitors as a new class of pharmacologic agents, of possible use in antitumor therapy, or in other diseases dominated by abnormal neovascularization. XI. Role of Angiogenesis in Clinical Oncology

There are a variety of seemingly unconnected clinical observations that may now be better understood because they are based on angiogenic phenomena. For example, many human tumors seem to exist at first in a prevascular state. An in situ carcinoma may stay for years at a small size of a few millimeters. Examples are carcinoma in situ of the bladder, breast, and cervix (Farrow et al., 1977; Hicks, 1977; Stafl and Mattingly, 1975). The onset of vascularization of a tumor marks the transition to more rapid growth, local invasion, and distant metastasis. The progression of superficial melanoma, for example Clark level I, to its more invasive, faster growing

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counterpart also correlates with the arrival of new blood vessels in the tumor. Another example of this transition is observed in ovarian carcinoma that has metastasized to the peritoneal lining. Tiny peritoneal implants may remain white, avascular, and fairly uniform in size until new capillaries penetrate from beneath the peritoneum. The implants then grow and ascites fluid in the abdomen becomes bloody. In fact, bleeding, when it represents a clinical sign of malignancy, generally indicates that a tumor no matter where it is located has become vascularized. Sometimes a vascularized tumor may be revealed because it can stimulate angiogenesis in a remote location. For example, neovascularization of the iris is commonly associated with neoplasms of the retina, especially the retinoblastoma. Another example is the appearance of neovascularization in an old mastectomy scar that precedes the recurrence of tumor beneath the scar. Metastasis is also influenced by angiogenesis. Prior to vascularization, tumors are generally unable to shed cells into the circulation. For this reason, prevascular tumors have a low probability of metastasizing compared to their vascularized counterparts. For example, melanomas that are less than 0.7 mm thick reside entirely above the basement membrane and are avascular. They are rarely, if ever, associated with metastasis (Seigler and Setter, 1977). As a tumor becomes vascularized, the number of cells released into the circulation correlates with the density of blood vessels in the primary tumor. Thus, in experimental animals Liotta et al. (1976)found malignant cells in the effluent of tumors implanted in the mouse thigh, but only after new blood vessels had appeared in the tumor. The number of cells shed from the primary tumor correlated with the density of tumor blood vessels and with the number of lung metastases observed later. Another clinical phenomenon probably related to angiogenesis is the breakdown of the blood-brain barrier observed in primary tumors of the brain as well as in metastases. The protein leakage and local edema are thought to be due to the uniquely permeable ultrastructure of tumor capillaries (Long, 1979). These capillaries tend to remain undifferentiated and immature and generally lack smooth muscle cells (Ausprunk and Folkman, 1977). For this reason the vascular bed of a large tumor may fail to respond to epinephrine or other vasoconstrictors; it is sometimes difficult to control bleeding from a large tumor bed at the time of surgery. Tumor dormancy is another clinical phenomenon in which angiogenesis may play a role, although there is less evidence. For example, a metastasis may appear in the lung 5 years after removal of a rapidly growing Wilms’ tumor in a child. A metastasis may appear 15-20 years after removal of a primary breast cancer. Where were these tumor cells during the long quiescent period? The exact nature of this dormancy is one aspect of the meta-

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static process that is most obscure. Several hypotheses have been proposed. One is that tumor cells could be arrested in a subendothelial position. In the rabbit ear chamber, this has actually been observed; endothelial cells sealed over a gap after tumor cells had passed through it. Another possibility is that once tumor cells have escaped from the vascular lumen, they might grow to a small spheroidal mass of a few millimeters, but remain in the prevascular phase for a prolonged period. During this period, they might, for various reasons, be unable to stimulate angiogenesis and thus a small population of tumor cells could lie dormant even though the cells themselves had not stopped proliferating. This is an area where speculation is plentiful but where experimental data would be most welcome. XII. Summary

The hypothesis that tumors are angiogenesis dependent has, in the past decade, generated new investigations designed to elucidate the mechanism of angiogenesis itself. Many laboratories are now engaged in this pursuit. Some are studying angiogenesis that occurs in physiological situations, whereas others are interested in angiogenesis that dominates pathological conditions. These efforts have led to (1)the development of bioassays for angiogenesis; (2) the partial purification and, in one case, the complete purification of angiogenic factors from neoplastic and non-neoplastic cells; (3)the development of new polymer technology for the sustained release of these factors and other macromolecules in uiuo; (4)the cloning and long-term culture of capillary endothelial cells; (5)the demonstration of the role of nonendothelial cells, such as mast cells in modulating angiogenesis; (6) the discovery of angiogenesis inhibitors; and (7)the demonstration that certain animal tumors will regress when angiogenesis is inhibited. The effects of angiogenesis inhibitors provide perhaps the most compelling evidence for the role of angiogenesis in tumor growth. It is conceivable that the original effort to understand the role of angiogenesis in tumor growth will also lead to the use of angiogenesis inhibitors as a new class of pharmacologic agents in a variety of non-neoplastic diseases such as arthritis, psoriasis, and ocular neovascularization. However, much work remains to be done before it will be possible to understand (1)the regulatory systems that govern capillary density in normal tissues; (2) the factors that maintain the viability of microvascular endothelium; (3)the development of the vascular system itself; and (4)the mechanism by which vascular regression occurs, both in the embryo and in the postnatal organism. A knowledge of the mechanisms which underlie these normal processes may help to enlarge our comprehension of tumor angiogenesis.

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ACKNOWLEDGMENTS This work was supported by USPHS Grant RO1-CAI4019 from the National Cancer Institute, by a grant to Harvard University from the Monsanto Company, by USPHS Grants GM-25810 and EY04002 (to Robert Langer), and by contributions from the Franzheim synergy Trust.

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