Proc. Nati. Acad. Sci. USAVol. 86, pp. 2229-2233, April 1989Cell Biology

Transforming growth factor /3 reverses the glucocorticoid-inducedwound-healing deficit in rats: Possible regulation in macrophagesby platelet-derived growth factor

(procoflagen/chemotaxis/fibroblasts)

GLENN F. PIERCE*t*, THOMAS A. MUSTOE§, JANE LINGELBACHt, VICTORIA R. MASAKOWSKIt¶PEGGY GRAMATESt§, AND THOMAS F. DEUELt¶Departments of *Pathology, tMedicine, §Surgery, and IBiological Chemistry, Jewish Hospital at Washington University Medical Center,Saint Louis, MO 63110

Communicated by David M. Kipnis, August 22, 1988

ABSTRACT Transforming growth factor ,B (TGF-fi) andthe platelet-derived growth factor (PDGF) are potent mitogenicpolypeptides which enhance rates of wound healing in exper-imental animals; in contrast, glucocorticoids inhibit woundrepair. The potential of TGF-fi and PDGF to reverse thisinhibition in healing was tested in methylprednisolone-treatedrats with deficits in skin wound strength of 50%. Singleapplications of TGF-fi (10-40 pmol per wound, 0.25-1 ,ug)applied locally at the time of wounding fully reversed thisdeficit in a concentration-dependent and highly reproduciblemanner. Wounds in glucocorticoid-treated animals were char-acterized by a near total absence of neutrophils and macro-phages and by a delayed influx and reduced density offibroblasts; however, such wounds treated with TGF-13 showedsignificant increases in wound fibroblasts and in intracellularprocollagen type I. PDGF did not reverse the deficit in woundbreaking strength in glucocorticoid-treated rats; there weremore fibroblasts in the PDGF-treated wounds, but thesefibroblasts lacked the enhanced expression of procollagen typeI found in TGF-,-treated wounds. The wound macrophages,required for normal tissue repair, remained absent from bothPDGF- and TGF-,B-treated wounds in glucocorticoid-treatedanimals. This result suggested that macrophages might nor-mally act as an intermediate in the induction of procollagensynthesis in fibroblasts of PDGF-treated wounds and thatTGF-,B might bypass the macrophage through its capacity tostimulate directly new synthesis of procollagen type I infibroblasts. Whereas PDGF does not stimulate procollagensynthesis, in a rodent macrophage cell line, PDGF induced ahighly significant, time-dependent enhancement of expressionof TGF-fi.

Polypeptide growth factors derived from platelets and in-flammatory cells are important mediators of tissue repairprocesses (1). Transforming growth factor f (TGF-f3) is ahighly conserved 25-kDa homodimeric polypeptide ubiqui-tously expressed by mammalian cells and recognized byhighly specific receptors on virtually every cell type analyzed(2, 3). TGF-,8 is released by cells that are localized at sites oftissue repair, such as platelets, activated macrophages,lymphocytes, and possibly fibroblasts (2-4). TGF-f3 is also apotent chemotactic agent for monocytes and fibroblasts (5,6); it induces the synthesis of extracellular matrix proteins byfibroblasts (7-10), and it induces reversible fibroplasia in vivo(11). TGF-,8 has been found to directly accelerate the rate ofhealing of linear incisional wounds in rats (12, 13).Another polypeptide growth factor, platelet-derived

growth factor (PDGF), is also a candidate wound hormone

(1). It is released by activated platelets, macrophages, andfibroblasts. PDGF has potent chemotactic activity for neu-trophils, monocytes, fibroblasts, and smooth muscle cells(14-16). PDGF stimulates these cell types to express activ-ities associated with inflammation and repair (17-19), recruitsinflammatory cells and fibroblasts in vivo, and accelerates thehealing of linear incisions in rats (20).

Glucocorticoids, in contrast, inhibit wound healing inhumans and in animal models of tissue repair (21, 22). Theinhibition of procollagen synthesis in fibroblasts and thedecrease in circulating monocyte levels associated withsystemic administration of glucocorticoids sharply reducethe potential host responses for wound repair in vivo (21-25).

In the present experiments, the potential roles of TGF-/3and PDGF in reversing the glucocorticoid-induced deficit inwound strength of rat linear skin incisions have been inves-tigated.

METHODSStudy Design and Model. A full-thickness linear skin

incision model in young adult male Sprague-Dawley rats(Sasco, Omaha, NB) was analyzed as described (12). Fromeach rat, two to three pairs ofdorsal skin strips were obtainedfor tensometry analysis, and one to two paired samples wereobtained for histologic analysis. Methylprednisolone acetate(Depomedrol, Upjohn) was injected intramuscularly into rats2 days prior to wounding. Rats were weighed at times ofinjection, wounding, and excision. Complete blood countswere analyzed by using a Coulter S Plus 4 automatedanalyzer. Highly purified human platelet-derived TGF-,f1(the kind gift of M. Sporn, National Cancer Institute) (2) andrPDGF-B, the recombinant homodimeric product of thehuman c-sis gene (the kind gift of A. Thomason, Amgen,Thousand Oaks, CA) were applied as described (12). Colla-gen alone (control wounds) did not influence the rate ofhealing (12, 13).Data Analysis. Eight rats were used for each variable unless

otherwise stated. Analyses of variance and paired t tests ofbreaking strength scores and of differences between matchedexperimental and control values were performed using theSAS data analysis system (Division of Biostatistics, Wash-ington Univ.). Breaking strength measurements (grams offorce; 1 g = 9.8 millinewtons) were performed on a tensiom-eter (Tensiometer 10, Monsanto) blindly on coded samples.

Histologic Analysis and Immunoperoxidase Staining.Matched paired samples of wounds were placed immediately

Abbreviations: rPDGF-B, the recombinant-DNA-produced B-B ho-modimeric form of platelet-derived growth factor; TGF-,B, trans-forming growth factor (3.tPresent address: Amgen, 1900 Oak Terrace Lane, Thousand Oaks,CA 91320.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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into acetic acid/ethanol (1:99, vol/vol) fixative and pro-cessed as described (20). Paired sections were microscopi-cally analyzed by two independent observers who did notknow which wound treatment the samples represented, usingan arbitrary scale from 0 to 4 for degree of cellularity and forcell types. Immunoperoxidase staining of samples was per-formed by established methods, using rabbit anti-rat procol-lagen type I monospecific antiserum (a generous gift of K.Cutroneo, Univ. of Vermont) at 2 gg/ml (24) and biotin-conjugated goat anti-rabbit IgG (Bethesda Research Labora-tories) followed by streptavidin-horseradish peroxidase (Be-thesda Research Laboratories) (26). Negative controls con-sisted of parallel sections incubated with comparable dilu-tions of irrelevant primary antisera. Paired wound sectionswere analyzed microscopically by two independent observ-ers as described above.RNA Isolation and Northern Blot Analysis. The BALB/c

murine macrophage cell line J774A.1 (American Type Cul-ture Collection) was treated with rPDGF-B at 50 ng/ml for upto 52 hr. Total cellular RNA was isolated by a modifiedguanidine hydrochloride procedure (27). Equal amounts (15tkg) of RNA were separated on standard denaturing formal-dehyde agarose gels, blotted onto nitrocellulose (Schleicher& Schuell), prehybridized for 2 hr at 420C, and transferred tofresh hybridization solution with the addition of 1 x 106cpm/ml of nick-translated TGF-P probe [1.6-kilobase (kb)insert of the sp65-Murf3a.s. plasmid, gift of R. Derynck,Genentech] labeled to a specific activity of 5 x 108 cpm//ug.After an overnight hybridization at 420C, the washed blot wasexposed to Kodak X-Omat film for 1 hr and quantitated on ascanning laser densitometer (LKB).

RESULTSEstablishment of Significant Impairment of Wound Healing

by Glucocorticoid Administration. Previous experiments haveshown that growth factors increase by 2-fold the forcerequired to rupture incisional wounds in normal rats, therebyaccelerating wound healing by nearly 3 days in the first weekof wounding (12, 20). However, in severely impaired woundhealing, the effects of growth factors have not been studied.A reproducible and significant inhibition of wound healingwas established by testing increasing doses of methylpred-nisolone (0, 12, 30, and 60 mg/kg) in a slow release vehicleto groups of four rats by a single injection 2 days prior towounding. The breaking strength ofwounds was an accurateand highly reproducible measure of impairment in woundhealing. Methylprednisolone at 30 mg/kg decreased woundbreaking strength by 74% and 47% at days 5 and 7 afterwounding, respectively. Higher doses of methylprednisoloneinduced too profound a reduction ofwound breaking strengthfor reproducible analyses.

TGF-13 Reverses Healing Impairment in Methylpred-nisolone-Treated Rats. Since previous investigations showedthat TGF-,B applied at the time ofwounding doubled breakingstrength of day 7 wounds in normal rats (12, 13), day 7 waschosen to measure the effect of increasing concentrations ofTGF-,B in glucocorticoid-treated rats (Fig. 1). A positivedose-dependent effect of TGF-f3 was observed on woundbreaking strength, beginning at 0.2 ,ug ofTGF-,B per incision.Full reversal of the wound-healing deficit resulted when 0.5-1 ,ug ofTGF-P per incision was applied. Wounds treated with1 Mg of TGF-P had breaking strength values nearly 200% ofcontrol values after 1 week (Fig. 1); the reversal of thewound-healing deficit was transient, however (Table 1).Statistically significant reversal was demonstrated at days 5and 7 after wounding but not at days 10-14. By this time, theinhibitory effects of the single pretreatment with methylpred-nisolone were diminished (Table 1) and, importantly, theaugmented healing in normal rats induced by TGF-f3 was

O GC* hTGF-p3 0.001

z 0.05

J'C 2000 0.01 0.5

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LU

1001

00 0 0.1 0 0.2 0 0.5 0 1.0 0 1.5

hTGF-P (,ug/incision)

FIG. 1. Dose-dependent increase in breaking strength of TGF-(3-treated wounds 7 days after wounding. Rats were injected with theglucocorticoid (GC) methylprednisolone (30 mg/kg) 2 days prior towounding. TGF-/3 was applied locally to one incision, at the time ofsurgery; the other incision served as a matched, paired control.Wounds excised on the seventh day showed a maximal TGF-/3 effectin normal rats (12). SEM and P relative to control are given above thebars.

declining (12). TGF-f3 thus fully reversed the deficit in woundhealing induced by glucocorticoids in rats.

Effects of rPDGF-B on the Methylprednisolone-InducedWound-Healing Deficit. rPDGF-B at 20 ug per incision wasineffective in augmenting the breaking strength of wounds inthe methylprednisolone-treated animals when tested at days 7and 10 after wounding (Table 1). In contrast, highly significantstimulatory effects ofrPDGF-B were reproducibly observed innormal rats at days 7 to 10 after wounding, enhancing thewound breaking strength to values 170% of control wounds(Table 1; ref. 20).

Analysis of Cellular Migration into Growth Factor-TreatedWounds. A striking influx of cells into TGF-p- and rPDGF-B-treated wounds in normal rats was observed previously(12, 13, 20). Histologically, these wounds resembled a highlyexaggerated normal inflammatory component of tissue re-

Table 1. Effect of TGF-,B or rPDGF-13 applied at the time ofwounding on the glucocorticoid-induced healing impairment

Day after Growth Breaking strength, gwounding factor TGF-.3-treated Control P

5 TGF-j3 49 ± 5 (42%) 34 ± 3 (29%7o) 0.017 TGF-f3 187 ± 22 (97%) 132 ± 20 (68%) 0.0210 TGF-,8 267 ± 25 (75%) 240 ± 24 (68%) NS14 TGF-f3 523 ± 71 (92%) 474 ± 43 (84%) NS7 rPDGF-/3 124 ± 14 (74%) 118 ± 7 (701%)10 rPDGF-P 216 ± 18 (49%7b) 233 ± 24 (53%)

Groups of eight rats were injected with methylprednisolone at 30mg/kg 2 days before wounding and either 1 ,ug of TGF-f3 or thecollagen vehicle alone was applied opposite to paired incisions oneach rat. Wounds were tested for breaking strength on the days onwhich TGF-,f- or rPDGF-,8-treated wounds were previously shownto have significantly stronger breaking strength measurements com-pared to matched controls (12, 20). Results are presented as mean ±SEM. P is for two-tailed paired t test on glucocorticoid-treatedsamples, growth factor-treated vs. controls. NS, not significant. Thepercentage of the normal control wound breaking strength values foreach day is shown in parentheses. The breaking strength of normalwounds treated with 2 ,&g of TGF-f3 is 180 g at day 5 and 310 g at day7. The breaking strength of normal wounds treated with 20 jig ofrPDGF-B is 360 g at day 7.

2230 Cell Biology: Pierce et al.

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4 ~~~~~~~~~~~.05~ ~ ~ 0

1 2 3 5 7 10

DAYS POSTWOUNDING

FIG. 2. Cellular influx into wounds. Rats were pretreated with theglucocorticoid (GC), and either TGF-,B (1 ,ug) or rPDGF-B (20 Ag)was applied locally at the time of surgery. Paired wounds from fourto eight rats per time point were excised and two separate histologicsections were obtained and rated for cellularity on a scale from 0 to4 by two independent observers who did not know which treatmentsthe samples represented. A score of0 represented baseline cellularityin unwounded dermis, and a score of 4 represented the degree ofcellularity observed 2-3 days after wounding in normal (untreated)wounds. Results from both observers were averaged and analyzed.Although some neutrophils were observed in wounds at days 1 and2, macrophages were not observed, and the vast majority of theincreased cellular influx from day 3 onward was due to fibroblasts,which peaked at days 5-7 after wounding in glucocorticoid-treatedrats compared to days 3-5 after wounding in normal rats. BothrPDGF-B and TGF-,8 enhanced the degree of fibroblastic influx butdid not alter the delayed kinetics induced by glucocorticoid treat-ment. The fibroblastic influx induced by rPDGF-B was more sus-tained than that found in TGF-p8-treated wounds. In contrast,granulation tissue was increased only in TGF-p-treated wounds (Fig.3).

pair. The cellular influx correlated directly with the potentinfluence of growth factors as chemotactic factors in vitro.Normal wound healing is characterized by the time-dependent influx of neutrophils within the first day afterwounding, followed by an influx of macrophages within 2-3

-,. __

days of wounding, and by an influx of fibroblasts by days 3-4 (13, 20). In contrast, in methylprednisolone-treated ani-mals, the wounds contained few neutrophils at days 1 and 2and were essentially devoid of macrophages (Fig. 2). Fibro-blast influx was delayed until days 5-7 after wounding.TGF-f3 and rPDGF-B applied locally had no effect onrestoring neutrophil and macrophage influx; both growthfactors significantly augmented the number of fibroblastsentering wounds (PDGF-B > TGF-f8), though entry was stilldelayed (see Fig. 2 legend). TGF-/3-treated wounds fromglucocorticoid-treated animals had increased granulation tis-sue 7 days after wounding (Fig. 3A), compared to matchedcontrol wounds (Fig. 3B), whereas rPDGF-B-treated woundsdid not. Thus, in the wounds from PDGF-treated rats, themore enhanced fibroblastic response was associated with amarked reduction in the formation of granulation tissue.TGF-P- and rPDGF-B-treated wounds in glucocorticoid-

treated animals lacked the influx of monocytes and neutro-phils which is characteristic in wounds in nonglucocorticoid-treated animals. A drop in the leukocyte count (WBC)reproducibly occurred after methylprednisolone treatment(normal, 9820 cells per mm3 vs. 3200 cells per mm3 2 daysafter glucocorticoid treatment; P < 0.01). Monocyte countsdecreased to nearly undetectable levels within 1 day afterwounding (normal, 680 cells per mm3 vs. glucocorticoid-treated, 80 cells per mm3; P < 0.001) before returning over thenext week to nontreated control levels. The loss ofcirculatingleukocytes correlated directly with the lack of neutrophilsand tissue macrophages in wounds in glucocorticoid-treatedrats. Since the monocyte/macrophage, but not the neutro-phil, is required for normal incisional repair (21), these resultssuggested that TGF-13 may enhance wound healing in thismodel by a direct influence on the fibroblast; PDGF, incontrast, may require the macrophage to mediate a significantcomponent ofthe tissue repair activities it has in normal rats.Enhanced Procollagen Type I in TGF-i3-Treated Wounds. In

normal wounds, intracellular procollagen type I may bedetected as early as day 2 after wounding in fibroblastsmigrating into the wound (unpublished observations). How-ever, in control, TGF-.8-treated, and rPDGF-B-treatedwounds from methylprednisolone-treated animals, intracel-lular procollagen type I was not detected until day 5 afterwounding, correlating directly with delayed fibroblast influx

FIG. 3. Photomicrographsshowing fibroblast and granula-tion tissue content in TGF-P-treated (A) and matched, pairedcontrol (B) wounds after 7 days.

I6 gAt the time of surgery, TGF-,B, 1-ug, or vehicle alone was applied toincisions. Wounds were harvested

'* *;K $ s - and processed for histologic anal-ysis. (Original magnification,N i-t; b x 100; printed at x70). The arrowsdemarcate the boundaries of new

b -Xx ; _ n granulation tissue through the der-mis. Note the increased numbersof fibroblasts in TGF-f-treatedwounds compared to controls.rPDGF-B induced increased num-bers of wound fibroblasts withoutthe increased granulation tissueobserved in TGF-,3-treated wounds(data not shown).

Cell Biology: Pierce et A

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(Fig. 4). Enhanced numbers of fibroblasts were found inTGF-P- and in rPDGF-B-treated wounds, but at days 5 and7 approximately twice the number of fibroblasts synthesizingprocollagen type I were detected in TGF-p-treated wounds(Fig. 4A) but not in rPDGF-B-treated or control wounds (Fig.4B). Thus, increased fibroblast influx coupled with an acti-vated fibroblast phenotype containing intracellular procolla-gen type I correlated directly with increased breakingstrength in TGF-,f-treated wounds. Moreover, since onlyTGF-/3 directly stimulated procollagen type I synthesis, it ispossible that rPDGF-B mediated its positive influence onnormal wound healing through induction of TGF-,8 in mac-rophages.

Analysis of PDGF-Stimulated Expression of TGF-13 inMouse Macrophages. In view of the striking ability of PDGFto attract and activate macrophages to release other cyto-kines (reviewed in ref. 1), the failure ofPDGF to reverse thewound-healing deficit in the present model might be ac-

counted for by the observed absence of tissue macrophagesfound at the site of the incision. This possibility was directlytested in vitro by determining whether rPDGF-B wouldinduce the time-dependent expression of TGF-f3 in a well-characterized rodent macrophage line, J774A.1. A 1-hr au-toradiogram of a representative Northern blot (Fig. 5) re-vealed significant levels of hybridizable TGF-p mRNA inthese cells at 2.6 (major band) and 1.9 (kb) (minor band),corresponding to previously published size estimates ofTGF-13 mRNA in rodent cells (28). Highly significant aug-mentations of levels ofTGF-f3 mRNA were observed at 4 and9 hr of rPDGF-B treatment (Fig. 5, lanes 4 and 5). Thistime-dependent increase was shown by densitometric quan-titation to be between 3- and 6-fold in four different experi-ments.

DISCUSSIONThe single local application of a purified polypeptide growthfactor at the time of wounding fully reversed the impairedtissue repair characteristic of the glucocorticoid-stressed rat.The stimulatory effect ofTGF-j3 in reversing impaired healingwas dose- and time-dependent; 40 pmol (1 Ag) per incisionwas most effective; maximal effectiveness was reached 7days after wounding and treatment. Wound strength in

FIG. 4. Photomicrographsshowing fibroblast influx andprocollagen type I staining inTGF-fi (1 ,ug) (A) and matched,paired control (B) wounds 5 daysafter wounding. Both fibroblastcontent and procollagen type Istaining (brown) were assessed oncoded slides. (Original magnifica-tion, x200; printed at x 140).These slides showed the wounddermis at the level of the pannic-ulus carnosus and were repre-sentative of increased fibroblastcontent and increased procollagentype I staining observed in TGF-,B-treated wounds compared tocontrols.

TGF-j3-treated wounds was n200% of values from glucocor-ticoid-treated rats that did not receive TGF-f3; wound-healingenhancement occurred in the critical early phase, whensurgical wounds in compromised patients are most likely todehisce. In this model, increased wound strength during thefirst 10 weeks of healing is directly correlated to increased netcollagen synthesis (29).The antifibrotic actions of glucocorticoids are well estab-

lished and are thought to be due to inhibition of collagensynthesis within fibroblasts (24, 25). In both steroid-sensitiveand -resistant species, glucocorticoids induce a profoundmonocytopenia and failure of tissue macrophages to appearin detectable numbers in wounds associated with a delayedmigration of phenotypically activated fibroblasts (Figs. 1 and3) (21, 23). The influx of wound macrophages during the firstseveral days after wounding is an absolute requirement fornormal healing (21). In glucocorticoid-treated animals, theabsence of the enhanced cellularity normally associated withincisional wounds correlated with the impaired breakingstrength and reduced new collagen synthesis; these effects

treatment 0 -rPDGF-Btimehrs 0 .5 4 9 12 26 52

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2 3 4 5 6 7 8

FIG. 5. Autoradiogram of Northern blot of TGF-P mRNA fromrPDGF-B-treated J774A.1 cells. J774A.1 cells, grown to confluencyand serum-starved, were treated and incubated with rPDGF-B at 50ng/ml for up to 52 hr. Total cellular RNA was isolated from cellsamples at the times shown. A 1-hr exposure of the blot is shown;lane 1 represents a zero time control with carrier bovine serumalbumin at 10 .ug/ml in serum-free DMEM. An increase in TGF-f3mRNA can be seen in lanes 4 and 5, representing RNA isolated after4 and 9 hr of rPDGF-B treatment, respectively.

A.-¼h trAP Ap;-MO~~~ ~ ~ ~ ~ ~ ~~~-He~~~~~~~ -. -C

tve~lF- _ '~' ~* Mu;77 of4p

:* v9o- 1'wv<s4

.401X

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were reversed by TGF-,8, which normally is constitutivelyexpressed by monocytes but secreted only from activatedmacrophages (4). These results suggest that although bothTGF-f3 and PDGF augmented fibroblast influx into wounds,TGF-f3 may have acted directly to increase procollagen typeI synthesis (24, 25). In contrast, it seems likely that recruitedand activated macrophages in normal wounds secrete TGF-,8, which, in turn, rapidly induce procollagen type I synthesis.Apart from its potent macrophage chemoattractant andactivating properties, PDGF affects wound healing throughits stimulation of collagenase secretion from fibroblasts, aneffect more likely of importance in collagen remodeling laterin wound healing (19). In contrast to TGF-/3 (7-9), PDGF hasnot been shown capable of directly stimulating procollagentype I synthesis by fibroblasts in vitro (11), and thus it wouldnot be expected to directly antagonize a glucocorticoid-mediated suppression of procollagen synthesis.

Glucocorticoids induce many changes in animals which mayinfluence wound healing. TGF-f8 did not enhance wouitdhealing to levels observed in TGF-3-treated wounds in normalrats and thus TGF-,8 is unable to reverse all of the negativeinfluences in wound healing induced by glucocorticoids.The absence of wound macrophages and the diminished

and delayed influx of fibroblasts in glucocorticoid-treatedrats may therefore reduce sharply the potential of PDGF tostimulate wound healing in the glucocorticoid-treated animal.This interpretation is supported by data showing that rPDGF-B increased TQF-f8 mRNA in J774A. 1 macrophages (Fig. 5),as well as TGF-,3 and PDGF-A chain mRNA in fibroblasts invitro (unpublished results; see also ref. 30). Of note is therecent finding ofconstitutive PDGF-B expression by J774A. 1cells (31), which may in fact augment the constitutive TGF-,fexpression observed in the present studies through an auto-crine loop. In vivo, rPDGF-B is a more potent recruiter ofinflammatory cells and fibroblasts into wounds than is TGF-,3(Fig. 2 and unpublished results). Preliminary observationsindicate that rPDGF-B can stimulate the in situ synthesis ofTGF-p protein contained in wound mononuclear cells within2-4 days of wounding. Although macrophages release anumber of other growth factors and extracellular matrixproteins likely to be important for tissue repair (i.e., inter-leukin 1, fibronectin) (31, 32), exogenous TGF-,B may sub-stitute in part for the macrophage requirement of healingwounds (21, 33) through its capacity to directly stimulate thesynthesis of procollagen type I in fibroblasts. PDGF, in turn,may regulate wound repair by means of cell recruitment andstimulation of new growth factor synthesis. Purified growthfactors are not now used as therapy for healing-impairedincisional wounds, although in vitro analysis ofactivities suchas chemotaxis (14-16) and cellular activation (17-19) and invivo experiments in stressed animals using wound chambers(34, 35) all support the hypothesis that growth factors maynormally function to direct the complex pathways leading toa healed wound.

We thank J. Reed for excellent technical assistance, Dr. M. Spornfor a generous supply of purified human TGF-,B, Dr. A. Thomasonfor helpful discussions and purified rPDGF-B, Dr. K. Cutroneo fora generous supply of anti-procollagen antisera, Dr. R. Derynck forthe TGF-13 cDNA probe, and Drs. E. Crouch and J. Jeffrey forongoing interest and advice. This study was supported by NationalInstitutes of Health Grants HL14147 and HL31102.

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Cell Biology: Pierce et al.

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