how is blood vessel growth regulated in normal and ... · fig. 2. final stages of capillary...

[CANCER RESEARCH 46, 467-473, February 1986]

How Is Blood Vessel Growth Regulated in Normal and Neoplastia Tissue?-G. H. A. Clowes Memorial Award Lecture1

Judah FolkmanDepartments of Surgery and Anatomy. Harvard Medical School, and Children's Hospital, Boston, Massachusetts 02115

I am greatly honored to receive the 25th G. H. A. ClowesMemorial Award. I thank the Board of Directors and the AwardsCommittee of the American Association for Cancer Research fortheir confidence.

Isolated Perfused Organs

For a number of years our laboratory has been studying themechanism of angiogenesis, especially as it relates to the growthof solid tumors. This work originated from a single experimentthat Frederick Becker and I did in 1962 at the Naval ResearchInstitute in Bethesda. We were studying hemoglobin solutionsas potential substitutes for blood transfusions. We perfusedthese solutions through canine thyroid glands in isolated glasschambers. The glands survived for about 2 weeks. To determineif these isolated organs could support growth, we injected themwith mouse melanoma cells. Tiny tumors developed but stoppedgrowing at 1-2-mm diameter and never became vascularized(1). We learned later that endothelial cells swelled and could notproliferate in the presence of free hemoglobin solutions lackingplatelets (2). Thus, in the isolated organ experiments we witnessed the fortuitous failure of neovascularization. However, thetumors were not dead. When they were transplanted to theirhost mice, they rapidly vascularized and grew to more than 1cm3 (Fig. 1). This was one of the earliest experimental modelswhich suggested that when a tumor is held in the "prevascular"

state, tumor growth may be suppressed.

Hypothesis: The Relationship of Capillary Growth and TumorGrowth

A few years later, in 1972, when these experiments wereconsidered together with those of Algire er al. (3) and Tannock(4), we proposed this hypothesis: solid tumors are angiogenesisdependent (5). After subsequent modifications (6), this idea cannow be stated in its simplest terms. Once tumor take hasoccurred, every increase in tumor cell population must be preceded by an increase in new capillaries that converge upon thetumor. We knew rather soon that this hypothesis might beimportant, because of the unexpected hostility and ridicule thatit generated. We have since concluded that this idea may havebeen ahead of its time, because we still have all of the reprints.This idea also generated much new work. It meant that we wouldhave to learn in some detail how capillary growth is regulated ifwe were to attempt to inhibit tumor growth by inhibiting angiogenesis.

Received 8/29/85; accepted 10/11 /85.1Supported by USPHS Grant R01-CA37395 from the National Cancer Institute,

by the American Cancer Society, by a grant to Harvard University from theMonsanto Co.. and by contributions from the Franzheim synergy Trust. Presentedon May 24.1985, at the Seventy-sixth Annual Meeting of the American Association

for Cancer Research, Houston, Texas.

The hypothesis also led to the following questions: (a) why arecapillary endothelial cells normally so quiescent that turnover ismeasured in years (7); (b) how is normal angiogenesis so tightlyregulated that in females the brisk neovascularization that accompanies ovulation, and also repair of menstruation, is shut offafter 2 or 3 days; (c) why is it that males can go through theirentire life without angiogenesis, unless wounded; (d) how dotumors break through this apparent natural barrier to capillarygrowth and stimulate intense angiogenesis, continuously?

New Methods for the Study of Angiogenesis

The first problem that confronted us was how to attack thesequestions. When we began our studies, the phenomenon ofangiogenesis was relatively inaccessible. For example, if youwere to examine the fresh specimen of a carcinoma of thebronchus, neovascularization would be grossly visible on thesurface of the tumor. Histological sections would show capillariesand their endothelial cells intertwined with neoplastic cellsthroughout the tumor mass. However, histological sections alonerevealed little about the mechanism of angiogenesis. Therefore,several new methods had to be developed. The rabbit corneabecame the basis of one of these methods (8).

The cornea is avascular. A small pocket can be made in it anda tumor implant can be inserted while the rabbit is anesthetized.The tumor is separated from the vascular bed of the host. Newcapillary blood vessels will grow in a linear manner toward thetumor, and the rate of vessel growth can be measured. It is alsopossible to implant a biologically inert sustained release polymer(9) impregnated with tumor-derived angiogenic activity. New

capillaries will grow toward the polymer implant at a mean rateof about 0.2 mm/day. When the angiogenic stimulus is discontinued by removing the pellet, these capillary vessels regress andeventually disappear over a period of weeks (10).

Sequential Events of Capillary Growth

Dianna Ausprunk (11) studied these implants by serial electronmicroscopy and elucidated the sequential steps by which anindividual capillary grows. The general features of capillarygrowth are similar regardless of the source of the angiogenicstimulus. To summarize: (a) New capillaries arise from smallvenules which lack smooth muscle, (b) In the presence of anangiogenic stimulus such as a small tumor nidus, endothelialcells within a venule begin to degrade the vascular basementmembrane and protrude through the wall of the vessel. Themicroscopic bleeding during this process may contribute to thebleeding which is considered an early clinical sign of cancer. Thelaboratories of Rifkin (12), Liotta (13), and Madri (14) have studiedthe enzymatic mechanisms of this local proteolysis of the basement membrane and of the subsequent degradation of interstitial

CANCER RESEARCH VOL. 46 FEBRUARY 1986

467

Research. on September 23, 2020. © 1986 American Association for Cancercancerres.aacrjournals.org Downloaded from

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REGULATION OF BLOOD VESSEL GROWTH

matrix, as the endothelial cells begin to move toward an angi-

ogenic stimulus, (c) The locomotion of endothelial cells towardthe angiogenic stimulus is associated with their linear alignmentas they form a capillary sprout, (d) A lumen is formed in thesprout, (e) Endothelial proliferation takes place within the sproutbut not usually at its tip. (/) The tip of one sprout joins withanother to form a capillary loop through which blood begins toflow. Granulation tissue that can be seen in an open woundconsists of myriads of these capillary loops. New sprouts originate from each loop to form an entire capillary network, (g) Newbasement membrane is formed and microvascular pericytes areincorporated into it.

The tumor cells that induce these capillary loops then proceedto grow rapidly around the loops to form microscopic cylinders.Tannock (4) has shown that the radius of these cylinders doesnot usually exceed about 150-200 um, which approximates the

oxygen diffusion distance. Nicosia ef al. (15) have shown in someelegant experiments that tumor cells prefer to grow contiguousto the vascular basement membrane of capillaries that are cultured in plasma clots in vitro. The intense recruitment of newcapillaries by tumors can be better visualized by injecting acasting solution into the vasculature of an excised tumor andthen digesting away the tumor cells (Fig. 2). In some humantumors, the pattern of capillary induction and the cylindricalconfiguration of tumor cells around capillaries are grossly visiblewithout removal of the tumor cells, e.g., the "papillary" carcino

mas of the thyroid and bladder. It is of interest that some of thesequential steps in capillary growth are radioresistant. At leasttwo generations of capillary loops can form in the cornea afterexposure to 8000 rads (16). Blood flow begins, but the lack ofendothelial proliferation restricts further capillary growth.

From experiments such as these we began to perceive oftumors as continuously releasing diffusible angiogenic factorsthat could stimulate capillary growth over distances of 2-5 mm.

Subsequently, when we learned how to culture and clone capillary endothelial cells by feeding them tumor-conditioned mediumin gelatin-coated dishes (17), we saw that these cells could

recapitulate an entire capillary network in vitro, including hollowtubes and branches (18). It became clear that the vascular

Fig. 1. Diagram of isolated perfused rabbit thyroid gland. Melanomas growingin the gland have reached a limit of 1-2 mm diameter and are not vascularized.When these tiny tumors were transplanted to mice, the tumors became vascularizedand rapid growth ensued. From Perspectives in Biology and Medicine (28), withpermission of the publisher.

Fig. 2. Final stages of capillary formation. Each capillary loop has formed by theanastomosis of two sprouts. The capillary loops in this scanning electron micrograph are growing into a human carcinoma of the larynx. Original magnification, x192. From Miodonski et al. (41), with permission of the publisher.

endothelial cell, after receiving an angiogenic signal, can expressa defined program leading to the generation of new capillaryblood vessels.

Role of Mast Cells and Heparin in Angiogenesis

But could this program be turned only "on" or "off," or could it

be modulated, i.e., amplified or suppressed? To borrow a lessonfrom immunology, are there helper cells for endothelial cells? Wedirected our attention to mast cells because they (and macrophages) are among the two cell types most commonly attractedto a wide variety of tumors. David Kessler and I (19) implantedtumors in the chick embryo and found a 40-fold increase in mastcells around a tumor implant before new capillaries arrived.Subsequently, Richard Azizkhan and Bruce Zetter in our laboratory found that a mast cell lysate or mast cell-conditioned

medium could stimulate locomotion (20) of capillary endothelialcells in vitro (21). Of all the mast cell products which were tested,only heparin could stimulate locomotion of capillary endothelialcells and substitute for the mast cell lysate or conditioned medium (21). When small quantities (6-25 ^9) of heparin were

added to the chick embryo, the rate of capillary growth (stimulated by tumor extract) was significantly increased (22). A non-anticoagulant fraction of heparin obtained from Robert Rosenberg (23) had a similar effect. From these experiments we beganto realize that some part of the heparin molecule was acting asa positive regulator of angiogenesis. It is important to recall thatcommercial heparin is not a pure compound but rather a heterogeneous preparation of hundreds of isomers with a molecularweight of approximately 16,000 to 35,000.

Affinity of Endothelial Cell Growth Factors for Heparin

Why should heparin behave as a mediator of angiogenesis? Ithad been shown that heparin could bind avidly to endothelialcells, i.e., approximately 106 heparin molecules per vascular

endothelial cell (24). Also, we knew from the studies of Karnovskyand Rosenberg (25) that vascular endothelial cells can producethe heparin and heparin-like molecules that are found on the

endothelial cell surface. Gradually the following idea occurred to


468




Michael Klagsbrun in our laboratory after discussions with YuenShing. Could heparin sitting on the endothelial cell surface insome way latch onto growth factors for endothelial cells andfacilitate the binding or uptake of these mitogens to the endothelial cell (Fig. 3)?

Purification of a Tumor-derived Angiogenesis Factor by Hep

arin Affinity

This idea, although untestable at the time, led to a method forthe complete purification of a tumor-derived angiogenic factor

that was also an endothelial mitogen (26, 27). We had beenattempting to purify a tumor-derived angiogenic factor for more

than 10 years. The work was frustrating. Our bioassays wereslow and consumed large quantities of tumor-derived fractions.

The purification procedure required multiple steps and recoverywas low. However, in the past 3 years Klagsbrun and YuenShing had made considerable progress working with a rat chon-

drosarcoma. A breakthrough in the purification effort occurredearly in 1983 when they used heparin-Sepharose chromatogra-phy. They were able to obtain a 500,000-fold purification to asingle band preparation on silver-stained sodium dodecyl sulfate-

polyacrylamide gel (26, 27). Only two steps were required,cationic exchange on Bio-Rex 70 followed by heparin affinity

chromatography. The purified factor is a cationic polypeptidewith an isoelectric point of about 9.8 and a molecular weight ofapproximately 18,000 (Fig. 4). It stimulates capillary endothelialcell proliferation in vitro half maximally at 1 ng/ml and stronglystimulates neovascularization in the chick embryo within 24 h atdoses of 100 to 120 ng. (For a detailed review of the reportedwork that preceded this finding see Ref. 28.) This factor hadanother unexpected effect. It transformed many critics into competitors.

Since the publication of this method last year (27), other knownendothelial cell growth factors have been purified to homogeneityby heparin affinity chromatography. These include brain-derived

basic fibroblast growth factor, purified by Gospodarowicz andassociates (29); basic and acidic fibroblast growth factors purifiedby Lobb and Fett (30); and cartilage-derived growth factor purified by Sullivan and Klagsbrun (31). Heparin-affinity chromatog

raphy has also been used by Maciag ef al. to partially purifyendothelial cell growth factor (32), and by D'Amore and Klags

brun to partially purify retinal-derived growth factor. An angi-

ENDOTHELIALâ€”GROWTH FACTOR

(Angiogenic)

Fig. 3. Diagram of the concept proposed by Klagsbrun and Shing (27) in ourlaboratory, that heparin or fragments of heparin on the surface of vascular endothelial cells may selectively bind endothelial cell mitogens that are also angiogenic.The symbols for the endothelial growth factor are drawn to represent the putativebinding site of the polypeptide and not the entire growth factor.

43K-J

25.7K-1

I 8.4 K-(

I4.3K-I6.2K-I

x 4.5HorLUCD5 4.3H

4.Htilo

3.9-om

I 2SLOT

NUMBER

.10 ,0 P. 00Â» 8 t in

I U I

4 8 12 16

FRACTION NUMBER

20

Fig. 4. Purified endothelial growth factor that is also angiogenic. derived fromrat chondrosarcoma as described by Shing ef al. (27). Chondrosarcoma extracellular matrix was digested with collagenase and purified by Bio-Rex 70 chromatography followed by heparin-Sepharose chromatography. On the left sodium dodecylsulfate-polyacrylamide gel electrophoresis stained with silver. K, molecular weightin thousands. Slot 1, molecular weight markers Slot 2, peak fraction of growthactivity in a single band (200 ng = 1000 units) This purified growth factor wasadded to cultures of cloned bovine capillary endothelial cells (BCÂ£).Half-maximalstimulation is induced by growth factor concentrations of about 1 ng/ml. Therecovery of activity from the tumor is about 5% and the yield of pure growth factoris about 1 ng from 5 g of crude chondrosarcoma matrix. Reprinted from Advancesin Cancer Research (42). with permission of the publisher

ogenic endothelial cell mitogen similar to that derived from therat chondrosarcoma has recently been purified to homogeneityfrom human hepatoma by Klagsbrun.2

All of these factors stimulate proliferation of endothelial cellsin vitro and induce angiogenesis in vivo. They all seem to sharethe property of strong affinity for heparin. This affinity seems tobe specific for heparin. For example, the chondrosarcoma-de-

rived growth factor and its normal counterpart from cartilage donot bind to other highly anionic glycosaminoglycans such aschondroitin sulfate and hyaluronic acid (27). The binding of thesefactors to heparin also seems to be independent of charge (Fig.5). Thus, platelet-derived growth factor, which is cationic and

with an isoelectric point, 9.8, that is nearly identical to that ofchondrosarcoma-derived growth factor and cartilage-derived

growth factor, binds only weakly to heparin and is not anendothelial cell mitogen. Furthermore, retina-derived growth fac

tor and endothelial cell growth factor, which are both anionic (piof about 5), bind tightly to heparin, while epidermal growth factor,which has an isoelectric point of 4.7 and is not an endothelialmitogen, does not bind to heparin.

Klagsbrun and Shing (34) have found it useful to classify thesefunctionally related polypeptides as "heparin-binding growth factors." It is too early to say if the structures of these factors are

closely related, because no amino acid sequences have beenreported at this writing (see "Note Added in Proof"). However,

there do not seem to be any inherent functional differencesbetween tumor-derived endothelial cell mitogens and those found

in normal tissues. We speculate that many normal tissues maycontain endothelial growth factors but that these factors are notusually expressed except under a tightly regulated program, asfor example during the brief angiogenesis that accompaniesovulation or wound healing. In contrast, tumors appear to continuously express angiogenic factors. Thus, a major distinctionbetween physiological angiogenesis and tumor angiogenesis

2M. Klagsbrunet al., unpublisheddata.


469




Ã•'FGF(96>CDGFI98)

J'ChDGF(98>xfHDGF-Cotionlc (6)

Ã•xIlDGFIS)HDGF-Anlonlc (51

xf PDSFH8)

-30

28262.42.22.0i.s1.61.41.21.00.80.60.40.20

Sample GradientELUTION POSITION FROM HEPARIN-SEPHAROSE

Fig. 5. Elution of growth factors from heparin-Sepharose. Factors with highaffinity for heparin (i.e., requiring ^ .0-1.8 M NaCI for elution) are endothelial mitogensand also angiogenic. Epidermal growth factor (EGF), which does not bind to heparin,and platelet-derived growth factor (PDGF), which binds weakly, do not stimulateproliferation of vascular endothelial cells. This idea was first reported by Shing e(a/. (27). FGF, fibroblast growth factor; CDGF, cartilage-derived growth factor;ChDGF, chondrosarcoma-derived growth factor; RDGF, retina-derived growth factor; HDGF, hepatoma-derived growth factor.

may be a temporal difference in the expression of endothelialgrowth factors.

We do not know what part of the heparin molecule is responsible for its ability to bind endothelial mitogens. This question isunder study. It is also not clear whether the angiogenesis-

promoting activity of heparin is due to the same putative component of heparin that binds endothelial cell mitogens. However,we think that it is prudent to try to identify the minimum fragmentof heparin that appears to act as a positive regulator of endothelial proliferation and capillary growth, because of a recent findingin our laboratory that a hexasaccharide fragment of heparin,which has little or no biological activity by itself, becomes anegative regulator of angiogenesis in the presence of a cortico-

steroid.

Inhibition of Angiogenesis by Heparin and a Steroid

This finding came about in the following way. We had begunto use the chorioallantoic membrane of the chick embryo todetect angiogenesis activity in fractions being purified from tumorextracts. This bioassay was more economic than the rabbitcornea. A window was made in the eggshell and the test fractionwas applied to the chorioallantoic membrane (35). The additionof heparin increased the speed of development of the angiogenicreaction so that it could be read 1-2 days later (22). But oneproblem with this assay is that occasionally eggshell dust fallson the chorioallantoic membrane and causes background inflammation. We guessed that adding cortisone to the chorioallantoicmembrane might eliminate the irritation from the shell dust butnot abolish the tumor-angiogenic reaction (because tumor angiogenesis in mice and rabbits had not been previously inhibited bycortisone). As expected, cortisone alone prevented shell dustinflammation without interfering with angiogenesis induced bytumor extracts. The surprise was that when heparin and cortisone were added together tumor angiogenesis was inhibited(36). Furthermore, when this combination of heparin and steroidwas suspended in a methylcellulose disc (2 mm diameter) and

implanted on the young (6-day) chorioallantoic membrane, grow

ing capillaries regressed leaving in their place, 48 h later, anavascular zone up to 4 mm in diameter. The antiangiogenic effectwas specific for growing capillaries. Mature nongrowing capillaries in older membranes were unaffected. Non-anticoagulant hep

arin had the same effect.Again we faced the question of whether a component or

fragment of heparin could be identified that was responsible forthis antiangiogenic activity. We realized that the appearance ofan avascular zone on the young chorioallantoic membrane couldbe the basis of an assay for angiogenesis inhibition, analogousto penicillin assays on an agar dish. Robert Langer and hisassociates (36, 37) produced a series of heparin fragments bydegrading heparin with heparinase which was catalytically pure.The heparin fragments were tested in the presence of hydrocortisone in shell-less chick embryos cultured in Retri dishes (36)

(Fig. 6). This simplified the reading of avascular zones andavoided the shell dust problem. Of the heparin fragments whichranged from oligosaccharides and octasaccharides down todisaccharides, a hexasaccharide fragment with a molecularweight of approximately 1600 was found to be the most potentinhibitor of angiogenesis (in the presence of a corticosteroid).

Fig. 6. A, 6-day-old chick embryo in Petri dish. Methylcellulose about to beimplanted on the chorioallantoic membrane. The disc contains heparin (50 ng/10jjl) and 11Â«-epicortisol (50 >ig/10 ^l). B. avascular zone that appears 48 h later(India ink injection of chorioallantoic membrane). Reprinted from Advances in CancerResearch (42), with permission of the publisher.


470




The combination of the heparin hexasaccharide fragment andcortisone also inhibited tumor-induced angiogenesis in the rabbit

cornea.The hexasaccharide fragment also brought about regression

of reticulum cell sarcoma in mice in which treatment was notinitiated until tumors were palpable (200-300 mm3), but a hexa

saccharide requirement of 7 mg/kg/day, twice daily, was beyondthe production capacity of our laboratory, especially if we wishedto treat enough mice for statistical significance. Injection of wholeheparin and cortisone caused some tumor regressions but notbefore the mice suffered hemorrhages. Since we could not obtainenough hexasaccharide fragment for systemic therapy, wasthere a simple way to abrogate the anticoagulant effect ofheparin?

We knew from clinical experience that heparin taken p.o. doesnot cause anticoagulation. Therefore, heparin (Abbott Panheprin)was fed to tumor-bearing mice which also received cortisone.

Heparin fragments appeared in the bloodstream but there wasno anticoagulation (36). Reticulum cell sarcoma, Lewis lungcarcinoma, B-16 melanoma, and bladder carcinoma regresseoj

in mice that received the combination of drugs but there was notumor regression when either drug was administered alone.Some types of tumor did not regress. For example, there wereseveral types of methylcholanthrene-induced tumors for which

neither angiogenesis nor tumor growth was inhibited by whatseemed to be optimal doses of heparin and cortisone.

Another curious observation was that the minimum effectivedose for most of the responsive tumors was approximately 200units of heparin/ml drinking water. As the dose was increasedup to 1000 units/ml, tumor regression was more rapid. However,when heparin doses were increased further, i.e., to 2000-5000

units/ml, rapid tumor growth resumed. This result suggestedthat whole heparin might contain separate fragments of whichat least one could be responsible for promoting angiogenesisand another (or others) could be responsible for inhibiting angiogenesis in the presence of corticosteroids.

We were perplexed that heparins manufactured by differentprocesses and different companies revealed quite different an-

tiangiogenic activities despite similar anticoagulant activities. Abbott Panheprin had been used in our first experiments becauseit was available without a preservative. However, by late 1982this heparin became unavailable because Abbott had made anearlier decision to discontinue heparin production. We soonfound that heparins from many other manufacturers also inhibitedangiogenesis in the chick embryo and in the rabbit cornea, butnone was as potent as Abbott Panheprin, and some had to beapplied at 8-10 times the concentration of the Abbott prepara

tion. However, when given p.o., almost all the other heparinstested had to be given at such high concentrations that thepromoter activity was unmasked before tumor regression wasachieved. In fact the next most potent heparin (manufactured byHepar, Inc.) could inhibit only reticulum cell sarcoma. Otherheparins were generally ineffective against any of the tumors.This variability suggests the existence of a component of heparinwith a specific sequence that may be critical for inhibition ofangiogenesis. An alternative possibility is that some non-heparin

contaminant is responsible for the antiangiogenic effect of wholeheparin. We believe that possibility is ruled out by the recentdemonstration that a synthetic heparin pentasaccharide (38)

inhibits angiogenesis in the chick embryo in the presence of acorticosteroid (39, 43) (Fig. 7).

While these experiments demonstrated that small fragmentsof heparin can cross the intestine and provided a glimpse,however tantalizing, of the possible therapeutic potential ofangiogenesis inhibitors, they also convinced us that there couldbe little or no further progress in understanding the synergisticaction of heparin and steroids, unless the components of heparinresponsible for its angiogenic and antiangiogenic properties weredetermined.

These experiments also revealed another enigma. While cortisone and hydrocortisone inhibited angiogenesis (in the presenceof heparin), dexamethasone seemed to have little or no effect atconcentrations similar to hydrocortisone, yet dexamethasonehas 30 times the glucocorticoid activity of hydrocortisone. San-

dor Szabo suggested that this could mean that the antiangiogenic activity of hydrocortisone was independent of its glucocorticoid activity. This idea was tested with the compound 11Â«-

epicortisol which is the biologically inactive isomer of hydrocortisone. It differs from hydrocortisone only in that the 11-hydroxyl

group lies below the plane of the molecule, yet in the presenceof heparin, 11a-epicortisol inhibited angiogenesis in the chick

embryo. Subsequent experiments in our laboratory have shownthat there is a class of corticosteroids that have little or noglucocorticoid or mineralocorticoid activity and for which theability to inhibit angiogenesis in the presence of heparin is theprincipal activity (39,43). After an optimum heparin concentrationwas determined (Fig. 8), a dose dilution curve (1-200 /Â¿g)was

generated for the corticosteroids listed in Table 1. The end pointfor angiogenesis inhibition was an avascular zone of 4 mm orgreater when read 48 h after implantation of the test compound.At least 20 chick embryos were read for each steroid concentration, in addition to embryos implanted with the steroid alone,heparin alone, or heparin and hydrocortisone at an optimumconcentration. An index of antiangiogenic activity for each steroidwas determined from its dose-response curve. The percentage

of embryos with avascular zones at the most effective dose was

6 12 25 50 75

vg/10 ulFig. 7. Dose-response curve of a synthetic heparin pentasaccharide (Choay) in

the presence of hydrocortisone (50 Â¿ig/10M!)implanted on the 6-day chorioallantoicmembrane. Up to 17 embryos per column. Only avascular zones with diameters of4 mm or greater are recorded here. Neither pentasaccharide alone nor hydrocorti-sone alone produced avascular zones.


471



100coLUgNce<Ã%

AVASCULOÃo-1Â¡71p

12 5 25 50 100 150 200

CONCENTRATION, Â¡Â¡g/IO pg

Fig. 8. Comparison of different heparins in the presence of hydrocortisone (50; 20 embryos per column. Each tot of heparin must be tested before use.

When heparin is used locally on the chick embryo (or in the rabbit cornea), heparinfrom almost any manufacturer can be made effective by simply increasing theconcentration of the less active heparins. In contrast, when oral heparin is administered to achieve systemic antiangiogenesis against mouse tumors (36), very highdoses of heparin overcome the antiangiogenic effect and seem to potentiateangiogenesis. Abbott Panheprin (D) brought about tumor regression at a sufficientlylow dose to avoid this enhancement. Heparin from Hepar, Inc. (â€¢),was also ableto bring about tumor regression but only in reticulum cell sarcoma. (AbbottPanheprin is no longer being manufactured.) D, Sigma heparin.

divided by the most effective dose expressed in micromoles(Table 1). The results of these experiments suggest that theantiangiogenic activity of corticosteroids (in the presence ofheparin) is associated with the pregnane structure and is governed mainly by structural components on the "D" ring. The 4,5-double bond in the "A" ring and the 11-hydroxyl group on the"C" ring are not essential for antiangiogenic activity. However,

absence of the 17-hydroxyl and the presence of 20 and 21carbons on the "D" ring lead to successive reduction of antian

giogenic activity.By using these guidelines to structure-activity relationships,

synthetic steroids have been made that inhibit angiogenesis buthave no glucocorticoid or mineralocorticoid activity. For example,6Â«-fluoro-17,21 -dihydroxy-16/3-methylpregna-4,9(11 )-diene-3,20-dione (Upjohn) has approximately 9 times the antiangi

ogenic activity of hydrocortisone (in the presence of heparin).This compound has also been shown to inhibit tumor-induced

angiogenesis in the rabbit cornea in the presence of heparin.However, none of these steroids has as yet been tested bysystemic administration in tumor-bearing mice, mainly because

of the unavailability of a sufficiently potent heparin.The antiangiogenic activity of certain corticosteroids in the

presence of heparin is a newly discovered function, independentof glucocorticoid and mineralocorticoid activities. We refer tothese compounds as "angiostatic" steroids (43). Certain of the

angiostatic steroids such as tetrahydrocortisol are natural metabolites of cortisone previously thought to be biologically inactive (40). Because these metabolites circulate before being excreted in the urine and because heparin is found on the endothe-

lial cell surface, one can speculate that these natural substancesmay act together as physiological suppressors of capillarygrowth over a long period of time. It is conceivable that thissynergism is one of the humoral barriers that a tumor mustovercome before it can make the transition from a prevascularlesion to a vascularized neoplasm.

Table1Comparison of antiangiogenic index with glucocorticoid and mineralocorticoid

activity

Steroids were tested at concentrations of 1,5, 25,100, and 200 ng/10 Miin thepresence of heparin (Hepar) 50 pg/10Â¡Awith at least 20 embryos per concentration.The antiangiogenic index was determined as described in the text. From Science(43) with permission of the publisher.

StructureAntiangi- Glucc-ogenic corticc*index activity

Â«*"*

Hydrocortisone

11a-Epihydrocortisol

Cortexolone

HfOH 361 i i 11

-OH

HjCOH 102 O O

860"

17a-Hydroxyproges-terone

Corticosterone

397 O* 0a

.Â¿6Â®CO-OH

HjCOH 86 0.3 1.6

CO

Deoxycorticosterone

Progesterone

Testosterone

Estrone

Pregnenolone

62 10

OH

000

25 O O

13 O O

000

Conclusion

In my more grandiose moments, I think of a day to come whenit may be possible to use angiogenesis inhibitors as an adjunctto conventional therapy. Perhaps we will find out what preventsthe endothelial mitogens contained in normal tissues from stimulating endothelial proliferation under physiological conditionsand why it is that tumors seem to continuously release thesefactors. Hopefully, we may eventually learn enough about the

Tetrahydro S 704

â€¢-OH

* The glucocorticoid and mineralocorticoid activities of cortexolone and of 17,,-

hydroxyprogesterone are virtually absent.

472




angiogenic process so that other diseases dominated by abnormal angiogenesis, e.g., many types of ocular neovascularization,might be brought under control.

Acknowledgments

I thank Pauline Breen for typing the manuscript.

Note Added in Proof

The complete primary sequences of the basic pituitary fibroblast growth factor(44) and the acidic brain fibroblast growth factor (45) have recently been published.

References

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2. Gimbrone, M. A., Jr., Aster, R. H., Cotran, R. S., Corkery, J., Jandl, J. H., andFolkman, J. Preservation of vascular integrity in organs perfused in vitro witha platelet-rich medium. Nature (Lond.), 222: 33-36, 1969.

3. Algire, G. H., Chalktey, H. W., Legallais, F. Y., and Park, H. D. Vascularreactions of normal and malignant tumors in vivo. I. Vascular reactions of miceto wounds and to normal and neoplastic transplants. J. Nati. Cancer Inst, 6:73-85,1945.

4. Tannock. I. F. The relation between cell proliferation and the vascular systemin a transplanted mouse mammary tumour. Br. J. Cancer, 22: 258-273,1968.

5. Folkman, J. Anti-angiogenesis: new concept for therapy of solid tumors. Ann.Surg., 175: 409-416,1972.

6. Folkman, J., Cotran, R. S. Relation of vascular proliferation to tumor growth.Int. Rev. Exp. Pathol., 76: 207-248,1976.

7. Denekamp, J. Vasculature as a target for tumour therapy. Prog. Appi. Micro-cire., 4: 28-38, 1984.

8. Gimbrone, M. A., Jr., Cotran, R. S., Leapman, S. B., and Folkman, J. Tumorgrowth and neovascularization: an experimental model using the rabbit cornea.J. Nati. Cancer Inst., 52: 413-427,1974.

9. Langer, R., and Folkman, J. Polymers for the sustained release of proteinsand other macromolecules. Nature (Lond.), 263: 797-800, 1976.

10. Ausprunk, D. H., Falterman, K., and Folkman, J. The sequence of events inthe regression of cornea) capillaries. Lab. Invest., 38:284-294,1978.

11. Ausprunk, D. H., and Folkman, J. Migration and proliferation of endothelialcells in preformed and newly formed blood vessels during tumor angiogenesis.Microvasc. Res., 74: 53-65,1977.

12. Gross, J. L, Moscatelli, D., and Rifkin, D. B. Increased capillary endothelialcell protease activity in response to angiogenic stimuli in vitro. Proc. Nati.Acad. Sci. USA, 80: 2623-2637,1983.

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14. Madri, J. A., and Williams, S. K. Capillary endothelial cell cultures: phenotypicmodulation by matrix components. J. Cell Biol., 97:153-165,1983.

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17. Folkman, J., Haudenschild, C. C., and Zetter, B. Long-term culture of capillaryendothelial cells. Proc. Nati. Acad. Sci. USA, 76: 5217-5221,1979.

18. Folkman, J., and Haudenschild, C. Angiogenesis in vitro. Nature (Lond.), 288:551-556,1980.

19. Kessler, D. A., Langer, R. S., Ptess, N. A., and Folkman, J. Mast cells andtumor angiogenesis. Int. J. Cancer, 78: 703-709,1976.

20. Zetter, B. R. Migration of capillary endothelial cells is stimulated by tumour-derived factors. Nature (Lond.), 285: 41 -43.1980.

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retina and hypothalamus: biochemical and biological similarities. J. Cell Biol.,99. 1545-1549,1984.

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45. GimÃ©nez-Gallego,G., Rodkey, J., Bennett, C., Rios-Candelore, M., DiSalvo,J., and Thomas, K. Brain-derived acidic fibroblast growth factor: completeamino acid sequence and homotogies. Science, 230:1385-1388,1985.


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1986;46:467-473. Cancer Res Judah Folkman

G. H. A. Clowes Memorial Award Lecture−−Neoplastic Tissue? How Is Blood Vessel Growth Regulated in Normal and

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