[CANCER RESEARCH 49, 415-419, January IS, 1989]

Factors Influencing Blood Supply in Wound Granuloma Quantitated by a New inVivo TechniqueViswanath Mahadevan,1 Ian R. Hart, and Graham P. Lewis

Department of Pharmacology, Royal College of Surgeons of England, 35-43 Lincoln's Inn Fields, London WC2A 3PN /K M., G. P. L.J, and Biology of MetastasisLaboratory, Room 536, Imperial Cancer Research Fund, P. O. Box 123, Lincoln 's Inn Fields, London WC2A 3PX [V.M., ¡.R. HJ, United Kingdom

ABSTRACT

A new quantitative assay for measuring angiogenesis in a s.c. locatedsponge implant in rats is described. Using this model, which detectsneovascularization by measuring alterations in '"\c clearance, it has

been shown that the known angiogenic factors, transforming growth factora and tumor necrosis factor a, cause maximum vascularization of thesponge to occur by Day 11 postimplantation compared with Days 15 to17 in control animals. The monokine interleukin la is shown to bestrongly angiogenic, suggesting that more than one macrophage-derivedcytokine may be the active mediator in macrophage-induced angiogenesis.Extracellular matrix proteins appear to play a role in regulating theangiogenic response such that presoaking sponges in laminili (40 Mg/ml)or fibrinogen (500 ¿ig/ml)solutions induced a significant reduction in thetime taken to achieve maximum "'\o clearance values; no such enhance

ment of neovascularization was observed when sponges were presoakedin type IV collagen (100 MB/ml)solution. The assay described here, whichis reproducible, objective, and quantitative, should be of considerable usein elucidating the molecular basis of angiogenesis regulation.

INTRODUCTION

Angiogenesis, the growth of new capillaries, occurs in avariety of physiological and pathological situations. Experimental evidence points to angiogenesis being a crucial antecedentto the sustained growth of primary and metastatic neoplasms(1). The formation of new vessels appears to be a well-orchestrated process involving a complex interplay of many cell types,their products (including growth factors and extracellular matrix components), and certain other variables attributable to theinternal environment (2, 3). The outcome of this interplay isthe migration and proliferation of microvascular endothelialcells and their eventual organization into mature functioningcapillaries.

Many in vivo models have been developed for the assay ofangiogenesis but, apart from the rabbit corneal micropocketassay (4) and the chick chorioallantoic membrane assay (5), allsuffer from a lack of objectivity and reproducibility.

Recently we have developed a new, objective, and reproducible method of measuring angiogenesis in a sponge implant inrats (6). This method detects neovascularization of an implanted sponge by measuring l33Xe clearance as an indication

of blood flow. The development of blood flow must result fromthe ingrowth of new vessels into the inert sponge. Administration of endothelial cell growth supplement into the spongeaccelerated the development of new blood vessels, whereasprotamine delayed the onset of neovascularization, findingswhich are consistent with previous observations (7, 8). We havenow refined the original model and show here that recombinantIL-la2 is strongly angiogenic in this assay. We have also confirmed the ability of such known angiogenic factors as TGF-a

Received 6/7/88; revised 9/30/88; accepted 10/11/88.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed.2The abbreviations used are: IL-In, interleukin la; TGF-a, transforming

growth factor a; TNF-«,tumor necrosis factor a; PBS, phosphate-buffered saline:'33Xe, radioactive xenon solution.

(9) and TNF-a (10, 11) to induce neovascularization in thissystem. Our results show that more than one macrophage-derived cytokine may be the active mediator in macrophage-induced angiogenesis (3).

A histológica! perspective of the sequence of events leadingto capillary formation in the sponge was obtained by excisingsponges from animals at different time points following spongeimplantation and examining stained sections of this material.

Finally, based upon previous in vitro studies demonstratingthe influence of various extracellular matrix components uponendothelial cell behavior (12-16), we have shown in our modelthat coating the sponge with laminin or fibrinogen under sterileconditions prior to implantation significantly accelerates theonset of angiogenesis, whereas coating the sponge with eithertype IV collagen or bovine serum albumin solution under similar conditions has no such effect.

MATERIALS AND METHODS

Animals. Young adult male Wistar rats (weighing ISO to 210 g) wereused in all experiments and were obtained from the Imperial CancerResearch Fund animal breeding unit, Clare Hall Laboratories, SouthMimms, Herts, United Kingdom

Rats were anesthetized for all procedures by the i.m. injection ofHypnorm (0.315 mg/ml of fentanyl citrate and 10 mg/ml of fluanisone;Janssen Pharmaceuticals, Grove, Oxford, United Kingdom) at a doseof 0.5 ml/kg of body weight. The animals were housed individually inplastic cages. Food and water were available ad libitum, and a 12-hdark/light schedule was maintained.

Sponge Implants. Circular, polyether Polyurethane sponge discs(1.25-cm diameter x 0.6-cm thickness) (Vitafoam, Ltd., Manchester,United Kingdom) were used. To the center of each disc, one end of a1-cm-long polyethylene tube (1.4-mm internal diameter; Portex, Ltd.,Hythe, Kent, United Kingdom) was secured with two 5-0 silk suturesin such a way that the tube was perpendicular to the disc face. Thesponge discs with attached cannulae were sterilized as described earlier(6).

Chemicals and Reagents. '"Xe (specific activity, 370 MBq in 3 ml)

was supplied by Amersham International, Aylesbury, United Kingdom.TGF-a (rat) was obtained as 20 ¿¿gof powder from Peninsula

Laboratories, Inc., Belmont, CA.Recombinant human TNF-«(specific activity, 1 x 10s units/mg) was

the generous gift of Dr. L. Tavernier of Biogeni, Gent, Belgium.Recombinant human IL-la (specific activity, 2.1 x IO7 LAF units/

mg) was supplied by Hoffman La Roche, Inc., Nutley, NJ.Laminin (mouse, in Tris-buffered NaCl), type IV collagen (soluble,

from human placenta), and fibrinogen (Fraction I, from rat plasma)were all obtained from Sigma Chemical Company, Ltd., Poole, UnitedKingdom.

Technique of Sponge Implantation. Following induction of anesthesia,the hair on the dorsal side of the animal was shaved, and the skin wascleansed with 0.5% chlorhexidine in 70% alcohol (Hibitane; ICI, Ltd.,Macclesfield, United Kingdom). Using aseptic techniques, a 1-cm-longdorsal, midline, vertical skin incision was made 8 to 9 cm caudad tothe occipital ridge and a subcutaneous pouch was fashioned, cephaladto the skin incision, by gentle blunt dissection with a pair of curvedartery forceps. A sterile sponge disc with attached cannula was thenintroduced through the skin incision into the subcutaneous pouch, andthe cannula was exteriorized by pushing its free end vertically up

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through a needle puncture in the skin directly overlying the center ofthe sponge at a point 5 cm cephalad to the skin incision. This distancebetween the skin incision and the definitive position of the sponge discwas felt necessary to avoid the healing skin wound from influencingvascularization of the sponge (17). The exteriorized cannula wasplugged with a stopper, and the skin incision was sutured with twointerrupted 5-0 silk stitches.

HlciuilFlow Measurements. For blood flow measurements, the roomwas air conditioned at 31'C, and the animal was anesthetized. The

technique used for blood flow measurement was as described earlier(6). In brief, 10 n\ of '"Xe solution were injected into the sponge, the

cannula was plugged, and the animal was positioned so that the spongewas 1 cm directly below a -y-scintillation detector. The washout of

radioactivity was monitored every 40 s on a SR7 sealer rate meter(Nuclear Enterprises, Ltd., Reading, United Kingdom) connected inseries to the y detector and printed automatically on an ESP401 a-numcric and graphic printer (English Numbering Machines, Ltd., London, United Kingdom) coupled to the former. The clearance of '"Xe

at 9 min following injection was expressed as a percentage and calculated as follows.

of clearance of '"Xe at 9 min following injection =

100 x(count at 9 min - background read

(initial count - background reading)iding)!ing) J

20-

10 12 14 16 li 20 22 24 26

Sequential Histology. On each of the following fixed time pointspost-sponge implantation (Days 1, 2, 4, 6, 8, 10, 12, 14, and 16), atleast three animals were killed by carbon dioxide inhalation. Spongeswere excised by dissecting them free of overlying skin and surroundingfascia and fixed overnight in neutral buffered formalin, and S-^msections were stained with hematoxylin and eosin.

TGF-cr, Il.-ln, TNF-o, and Angiogenesis. Sterile sponges were implanted in 40 animals. TGF-«(3 jig in 10 M'of PBS) was injected intothe sponge in each of 5 animals on Day 6 postimplantation. TNF-awas prepared in 10 /il of PBS at 3 different concentrations: 5 ng/10 /il;50 ng/10 it\; and 5 ¿ig/10n\. On each of Days 6, 7, 8, and 9 postimplantation, groups of 5 animals received injections into the sponge ofone of these different concentrations. IL-1«was made up in 10 n\ ofPBS at 3 different concentrations: 0.48 ng/10 ^1; 4.8 ng/10 n\; and 48ng/10 fil. On each of Days 6. 7, 8, and 9 postimplantation, three groupsof 5 animals received injections of one or other of these concentrations.A control group of 5 animals each received 10 n\ of PBS into thesponge on Days 6, 7, 8, and 9 postimplantation.

Fibrinogen, Kxtracellular Matrix Proteins, and Angiogenesis. Sterilized sponge discs with attached cannulae were immersed for 4 h at25°Cin one of the following four sterile solutions, laminin in PBS (40

fig/ml), type IV collagen in PBS (100 ¿ig/ml),fibrinogen in PBS (500»igof flottable protein/ml), or bovine serum albumin in PBS (100 ¿*g/ml), and then implanted into five animals per group.

RESULTS

Standard Clearance. The combined results from a series ofexperiments in which 133Xeclearance from untreated sponges

was measured are presented in Fig. 1. For the first 2 to 11 daysafter implantation, clearance of '"Xe at 9 min after injection

varied between 11 and 22%, a value which represented clearanceby passive diffusion. From Day 11 more active clearance, representing the development of new blood flow, was evident. ByDays 15 to 16 after implantation, the 9-min clearance valueshad plateaued at around 50%, a figure equivalent to the 9-minclearance of s.c. injected l33Xe in the absence of sponge implantation (51 ±3.1%, n= 16).

Beyond Day 19, the clearance values fell sharply to return to11 to 22% by Days 21 to 25 (Fig. 1).

DAYS POST-IMPLANTATION

Fig. I. Standard curve of 9-min postinjection "3Xe clearance values. Points,

mean obtained from a minimum of seven animals: bars, SEM.

Histology. Representative photomicrographs depicting thehistological appearance of sponges 4, 8, and 12 days afterimplantation are given in Fig. 2. Initially the sponge material,which has a retractile, angular appearance, is separated by aleukocyte infiltrate and strands of fibrinous material; no bloodvessels are apparent (Fig. 2A). By 8 days after sponge implantation the interstices are filled with a more cellular infiltratecomposed of neutrophils (fewer in number than at Day 4),macrophages, fibroblasts, and endothelial cells lining new capillaries. A few fine collagen fibers are evident, but the generalappearance is one of disorganization (Fig. 2B). By Day 12postimplantation the rather disorganized appearance has givenway to one of reduced cellularity and increased collagen matrixdeposition, giving the field an appearance of greater organization. A phagocytic response to spicules of sponge is evident,and there are maturation and organization of the capillarynetwork with erythrocytes evident in the lumina of these newvessels (Fig. 2C).

TGF-a, TNF-a, IL-la, and Angiogenesis. Results from thisexperiment are shown in Table 1. Up to and including day 9postimplantation, there was no significant difference in theclearance values between the various groups (data not shown).On Day 11 postimplantation animals that had received either3 /ig of TGF-a, 5 ng of TNF-a, or the two higher doses of IL-la (4.8 ng and 48 ng, respectively) showed significantly higherclearance values than the control group (P < 0.001). In contrast,animals that had received the two higher doses of TNF-a (50ng and 5 ng) or the lowest dose of IL-la (0.48 ng) showed nodifference in clearance values compared with the control group(Table 1).

Extracellular Matrix Proteins and Angiogenesis. The resultsof this experiment are shown in Fig. 3. Up to and includingDay 7 postimplantation there was no significant difference inthe xenon clearance values among the 4 groups of animals.

On Day 9 postimplantation animals implanted with spongespresoaked in laminin or fibrinogen showed mean clearancevalues of 36.6 ±4.2 (n = 5) and 46.7 ±4.5 (n - 5), respectively,

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INDUCTION OF ANGIOGENESIS

'* . > .-

•¿�••¿�- :--.:-.-..

Fig. 2. Hematoxylin: eosin-stained 5-nm sections (X 100) throughsponges at 4 (I). 8 (//). and 12 (C) days postimplantation. In A, theretractile angular material is the polyether polyurethane sponge matrix. Interstitial space is occupied by fibrinous strands and a leukocyte,predominantly neutrophil, infíltrate. In lì.the space between thesponge spicules now is occupied by a disorganized accumulation ofneutrophils, macrophages, and fibroblasts. Immature capillaries linedby endothelial cells are evident, and some fine collagen fibers are justvisible in certain areas. In C, cellularity of the infiltrate has beenreduced, and the deposited collagen matrix is more apparent. Fibroblasts are organized, and there has been maturation of capillarieswhich now contain erythrocytes. A phagocytic response to the spongematerial has been mounted as indicated by the presence of a numberof foreign body-type giant cells.

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compared with the control animals which had received sponges By Day 12 postimplantation, the mean clearance value ofpresoaked in bovine serum albumin (20.1 ±2.3, n = 5). Thesedifferences were significant at P < 0.01 for the "laminin group"and P < 0.001 for the "fibrinogen group." No significantdifference was obtained between the "type IV collagen group"

and control.

59.4 ±3.5 (n = 5) from the laminin group was still significantlydifferent (P < 0.01) from control values [39.5 ±5.2 (n = 5)].However, by Day 14 postimplantation these differences ¡nclearance values among the groups had been abolished as allthe groups attained maximal vascularization (Fig. 3).

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INDUCTION OF ANG1OGENES1S

Table I Clearance values of ' "Xe injected into implanted sponges

Clearance of '"Xe(%)

Control animals"(5)*TNF-n!

5 n/(5)TNF-a, 50 ng (5)TNF-«,5.3n¿(S)IL-i,..

0.48 n/(5)IL-la, 4.8 ng/ (5)IL-1«,48ng7(5)Day

11postimplantation25.4

±1.0r56.9 ±1.6'49.1 ±4.3'

28.1 ±2.127.7±0.928.0

±5.049.5 ±0.8'49.2 ±4.0'Day

15postimplantation50.7

±2.845.4 ±2.750.4 ±4.549.5 ±5.247.2 ±3.540.0

±9.745.0 ±3.944.7 ±2.8

" Receiving injections of 10 pi of PBS on Days 6, 7, 8, and 9 postimplantalion.* Numbers in parentheses, number.' Mean ±SEM.

Injected with a single dose of TGF-a in a 10-pl volume on Day 6 postimplamation.

' Statistically significant (/' < 0.001) differences in mean value compared withcontrol values using Student's i test.

^Injected (with given dose in a 10-^1 volume) on Days 6, 7, 8, and 9 postimplantation.

80

60 -

I ii il \s *•IT

40 -

20-

I

12

DAYS POST-IMPLANTATION

Fig. 3. '"Xe clearance values 9 min postinjection. Columns, mean obtained

from five animals per group; bars, SEM. Sponges precoated with laminin solutionat 40 »ig/ml(•),fibrinogen solution at 500 Mg/ml (&), type IV collagen solutional 100 »jg/ml(•),or bovine serum albumin at 100 fig/ml (O) as detailed in text.

DISCUSSION

The technique used for determining angiogenesis in thepresent study appears to offer some significant advantages overexisting methods. The technique is based upon tìieassumptionthat new blood vessel formation in the sponge is responsiblefor alterations in vascular flow. In granulation tissue it ispossible that other factors, such as the pressure of extravascularfluid on the capillaries or the cellular composition of the granuloma, could also have some effect on local hemodynamics.However, within these limitations assessment of neovasculari-zation is objective compared with most methods which are, atleast in part, subjective (3). Continuous monitoring of the same

animal is possible because the injected l33Xe is cleared by

expiration within 2 h. Our decision to use and compare valuesat a single time point, rather than the more time-consumingderivation of clearance constants, was based on our earlierdemonstration of an excellent correlation coefficient betweenthese values and K¡values derived by curve peeling (18). Resultsobtained with this technique are very reproducible, as evidencedby the small standard errors, and this may reflect the minimalamount of disturbance or discomfort that sponge implantationproduces.

Sequential histology of sponges excised from animals atdifferent time points following implantation shows a cleartemporally ordered set of events. The early appearance ofneutrophils, followed by the appearance of macrophages, issucceeded by the appearance of fibroblasts and endothelial cells,the laying down of an amorphous extracellular matrix, and thesubsequent appearance and development of capillaries (Fig. 2).These capillaries were much in evidence histologically at Day19 postimplantation and later. The reason for the rapid declinein clearance values seen at this time point (Fig. 1) is notimmediately apparent. It may be that this change representsdiminished permeability of capillaries or thrombosis and cessation of flow, phenomena well documented in wound-healingcapillaries (19, 20). In light of this finding it should be notedthat a further advantage of the assay is that it reflects thefunctional state of new vessels rather than simply documentingtheir mere absence or presence. A single injection of TGF-a (3Mg)into the sponge, 6 days after sponge implantation, inducedfull vascularization by Day 11 compared with Day 15 in controlanimals (Table 1). TGF-a has been reported as being angiogenicat this dose in the hamster cheek pouch (9), a site which hasbeen criticized as perhaps being less sensitive than the cornea(3). As reported recently, TNF-a, which is a major secretoryproduct of activated macrophages, also is angiogenic (10, 11).In our hands, the enhancement of blood vessel growth was onlyseen after 4 daily injections of the lowest dose used in ourstudies of 5 ng. This concentration is comparable to thatreported to induce angiogenesis in the rat cornea and on thechick chorioallantoic membrane (10). The lack of a responsewith the higher doses of TNF-a, in contrast with the findingsin the rabbit cornea (11), may be because these higher dosesevoked intense inflammatory responses in the cornea but notthe sponge (11).

At the two higher doses of 4.8 ng and 48 ng, IL-la induceda strong angiogenic response, full vascularization being established by Day 11 versus Day 15 in control animals. Theseresults indicate that IL-la is a mediator of angiogenesis atconcentrations equivalent to or lower than those reported forother angiogenic peptides such as fibroblast growth factor,angiogenin, epidermal growth factor, or TGF-a (9, 11,21).

IL-1 is primarily a product of activated macrophages (21)and has a variety of effects including induction of T-cell, neu-trophil, and osteoclast activation; cartilage résorption;prosta-glandin and collagenase synthesis; cytotoxicity for tumor cells;antiviral activity and 0-interferon induction; pyrogenic activity,and activation of cultured human vascular endothelial cells toinduce a tissue factor-like procoagulant activity (22, 23). Themechanisms of action of IL-1 in inducing angiogenesis areunknown at the present time. However, this molecule, whichshows good homology to acidic fibroblast growth factor (24),could, when released from activated macrophages in concertwith TNF-a and other mediators, play a role in regulatingwound repair and in stimulating tumor development by enhancing the rate of growth of new blood vessels (3). A recent

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report also has suggested that IL-1 possesses angiogenic activity(25), and in our assay system, which responds well to theaddition of known angiogenic factors, we have confirmed thisobservation.

The crucial importance of a provisional stroma for the ingrowth of new vessels has been documented in several reports(for review, see Ref. 15). Based on evidence obtained from invitro studies it has been suggested that various components ofthe extracellular matrix, either singly or in combination, canmodify the proliferative and phenotypic behavior of microvas-cular endothelial cells (12-14). We now clearly show thatcoating sponges with laminin can accelerate significantly theonset of angiogenesis in vivo and confirm the likely role offibrin in stimulating this process (16). Given the recent observation that human endothelial cells express integrin-like proteins at their cell surface which mediate their attachment toadhesive proteins and the extracellular matrix (26), it is possiblethat provision of such substrates on the implanted spongesleads to interactions with these receptor molecules which facilitate the attachment and migration of infiltrating endothelialcells. Further studies involving coating the sponge with variouscombinations of matrix proteins and growth factors should helpto elucidate the molecular basis of some of the steps involvedin regulating angiogenesis.

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Benett, C., Rodkey, J., and Fitzpatrick, S. Pure brain-derived acidic fibroblastgrowth factor is a potent angiogenic vascular endothelial cell mitogen withsequence homology to interleukin 1. Proc. Nati. Acad. Sci. USA, 82: 6409-6413, 1985.

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1989;49:415-419. Cancer Res   Viswanath Mahadevan, Ian R. Hart and Graham P. Lewis 

Techniquein VivoQuantitated by a New Factors Influencing Blood Supply in Wound Granuloma

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