Gut 1996; 39: 172-175

Acceleration of wound healing in gastric ulcers bylocal injection of neutralising antibody totransforming growth factor 1

H Ernst, P Konturek, E G Hahn, T Brzozowski, S J Konturek

AbstractBackground-Application of neutralisingantibodies (NAs) to transforming growthfactor 1, (TGF1) improves wound heal-ing in experimental glomerulonephritisand dermal incision wounds. TGF1, hasbeen detected in the stomach, but despitethe fact that this cytokine plays a centralpart in wound healing no information isavailable to determine if modulation ofthe TGF1, profile influences the healing ofgastric ulcers. This study examinesgastric ulcer healing in the rat after localinjection ofNAs to TGFP1.Method-Chronic gastric ulcers wereinduced in Wistar rats by the application of100% acetic acid to the serosal surface ofthe stomach. Immediately after ulcerinduction and on day 2, NAs to TGF1(50 ,ug), TGF1, (50 ng), saline or controlantibodies (IgG; 50 Kug) were locallyinjected into the subserosa. Controlsreceived no subserosal injections. Animalswere killed on day 5 or 11, the ulcer areawas measured planimetrically, sectionswere embedded in paraffin wax, andstained with trichrome or haematoxylinand eosin. Depth of residual ulcer wasassessed on day 11 by a scale of 0-3, thepercentage of connective tissue was deter-mined by a semiquantitative matrix scoreand granulocytes and macrophages in theulcer bed were also assessed.Results-The application ofNAs to TGF,13led to a significant acceleration of gastriculcer healing on day 11 (0.6 (SD 0.8) v 3.7(SD 2.6) pn2), a reduction in macro-phages (23.7 (SD 22.6) v 38 (26) per 40Xpower field) and granulocytes (8.5 (SD 5.6)v 20 (10) per 40X power field), fewer histo-logical residual ulcers (mean 1 (SD 0.9) v 2(1.1)), a reduced matrix score, and aregenerative healing pattern. Excessivescarring was seen in the TGF13 treatedgroup.Conclusion-Further treatment of gastriculcers may induce a new treatmentmodality by local injection ofNA to TGF1,in an attempt to accelerate and improveulcer healing.(Gut 1996; 39: 172-175)

Keywords: gastric ulcer, wound healing, TGFI31,neutralising antibodies to TGF, 1, quality of woundhealing.

Fetal wounds heal with a reduced inflamma-tory and cytokine response and complete

restitution of the normal tissue architecture.1In contrast healing of adult wounds does notnecessarily lead to complete restitution ofnormal structure and function. Studies inhumans and animals have shown that patho-logical accumulation of extracellular matrix is acentral biological feature of poor healing andpoor preservation of the tissue architecture ininterstitial lung fibrosis, glomerulosclerosis,scarring of skin wounds, and healing of gastriculcers.2-7 Transforming growth factor 1(TGFI31) has been shown to act as a molecularswitch that turns the repair process on and offby exerting a variety of effects on the extracel-lular matrix. Data obtained from studies inanimals and humans show that TGFI31 plays acentral part among cytokines in the stimulationof matrix production, inhibition of matrixdegradation, and modulation of matrix recep-tors to increase cell adhesion to the matrix.3 8

In animal studies it has been shown thatseveral growth factors applied to gastric ulcers,such as epidermal growth factor, TGFox,platelet derived growth factor, and bFGF,accelerate the healing process by mechanismsthat are not completely understood.9-12TGF131 has been detected in the rat

stomach13 but no information is available atpresent, as to whether TGF13 has any influ-ence on the healing of gastric ulcers. In experi-mental glomerulonephritis and dermal incisionwounds, systemic application of neutralisingantibodies (NAs) to TGF,13 improved thehealing process and reduced the deposition ofextracellular matrix.3 6 Application of TGF13to skin wounds in rats resulted in an increase incollagen synthesis within the wound, andaccelerated rate of healing6 14 as with NAs. Inthis study we report that locally applied NAs toTGF13 in an experimental model of chronicgastric ulcer leads to an acceleration of ulcerhealing, a reduction in extracellular matrixdeposition, and an improvement in the restora-tion of tissue architecture.

Methods

Animal modelIn all the experiments, chronic gastric ulcerswere induced in male Wistar rats weighing150-180 g by the method of acetic acid appli-cation to the serosa described elsewhere."1Each group of animals contained 9-10 rats.The results were pooled for statistical analysis.

Experimental designTwo series of experiments were performed. In

Department ofMedicine, UniversityofErlangen-Nuremberg, Erlangen,GermanyH ErnstP KonturekE G Hahn

Institute of Physiology,Jagiellonian UniversitySchool ofMedicine,Krakow, PolandT BrzozowskiS J Konturek

Correspondence to:Dr H Ernst, Department ofMedicine IV, Charite,SchumannstraBle 20/21,10117 Berlin, Germany.Accepted for publication31 October 1995

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Acceleration of ulcer healing by application ofNAs to TGF,17

the first series 50 rats were divided into fivegroups and treated with local anti-transform-ing growth factor therapy or controls (asdescribed below), were killed after day 5, andthe ulcer size was measured. In the secondseries 50 rats were divided also into the samegroups and given the same treatments, butwere killed on day 11. Tissues were removedand the ulcer size was measured and the ulcerarea or scar was embedded in paraffin wax.

Local anti-transforming growth factor therapyImmediately after the induction of ulcers(during laparotomy) four of five groups, eachcontaining 10 animals, received local (inthe area of application of acetic acid) sub-serosal application of either 50 Kug NAto TGFI,I (AB-101-NA, R and D Systems,Minneapolis, USA), 50 ng TGFI3 (BDP1,British Biotechnology, Oxford), 50 Kugchicken IgG-control (AB 101-C, R and DSystems, Minneapolis, USA), or saline(0.9%). The fifth group received no sub-serosal application.On day 2 a laparotomy was performed and

the NAs or control antibodies (and saline)were again injected locally into the ulcer area.These subserosal injections comprised therespective substance in phosphate bufferedsaline at a volume of 100 ,ul, and were appliedjust beside the ulcer on the same wall of thestomach. In one group, only a laparotomy wasperformed (sham operation).

Specificity ofNAs to TGFJ,1The specificity of the neutralising antibodyagainst TGFP, has been tested in directELISA and western blot analysis by R and DSystems. This antibody shows 10% cross reac-tivity with TGF(5 and

Ernst, Konturek, Hahn, Brzozowski, Konturek

Figure 1: (A) Effect oftreatment with NAs toTGF,81, TGFl31, orcontrols on the healing ofchronic gastric ulcers onday 5 after induction of theulceration (n= 9-10 eachgroup). Results areexpressed as means (SD).Error bars= 1 SD. (B)Effect of treatment withNAs to TGFI31, TGFJ31,or controls on the healing ofchronic gastric ulcers onday 11 after induction ofthe ulceration (n= 9-10each group). Results areexpressed as means (SD).Asterisk shows significant(p*2 X Xcc

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T TT

cc>. LL

z- c (DCm._ 4.C.c

< _

Z

cm E P. U- W-.E X > c_ X

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'S'~~'gFigure 3: Gastric ulcer treated with TGF3, (trichromestain). Scarring in the submucosa (sm) (originalmagnification X25). Bar 100 um.

and fewer neutrophil granulocytes on day 11(8.5 (SD 5.6) v 20 (SD 10)) per low powerfield (X40 objective) than the controls. Thenumber of macrophages and granulocytes inthe ulcers treated with TGFI31 did not differsignificantly from those in the controls.

Figure 2: Gastric ulcer treated with NAs to TGFW1.Regeneration ofnormal tissue architecture. Newlyformedmuscularis propria (arrows; mp) and small submucosa(sm) (original magnification X25). Bar 100 ,um.

Histological examination (trichrome andhaematoxylin and eosin stain) of the connec-tive tissue compartment showed that ulcerstreated with NAs had a more regenerativepattern ofnormal gastric mucosal architecture.Collagen fibre orientation in the gastricmucosa showed an almost normal regenerativepattern. A semiquantitative assessment of con-nective tissue fibres in the ulcer bed was madeusing a semiquantitative matrix score. Thematrix score (mean ofNA treated group 19 v26 in the TGFI31 treated group, and a mean of23 in the control groups v 13 in normal sub-mucosa) showed a less dense distribution ofextracellular matrix in the submucosa of theulcer bed in the NA treated group (Fig 2). Thedifference of the matrix scores between theNA to TGF,B1 treated group and the TGF31treated group is statistically significant(p

Acceleration of ulcer healing by application ofNAs to TGF/31 175

promotes extracellular matrix deposition notonly by stimulation of extracellular matrixprotein synthesis, but also by inhibition ofprotease synthesis, stimulation of proteaseinhibitor synthesis, and an increased synthesisof cell adhesion receptors, which function tobind a wide variety of extracellular matrix com-ponents.2

In gastroduodenal ulcer disease poor healingwith submucosal scarring has been implicatedas a factor of local ulcer recurrence.7 Shiannet al have shown that different therapeutictreatments may lead to different patterns ofhistological maturity.7 Tarnawski et al foundthat treatment with Maalox led to better sub-mucosal healing and improved the overallquality of ulcer healing in contrast with treat-ment with H2 blockers.'5There is now evidence that overproduction

ofTGFf, may lead to scarring.5 6 14 16 In evo-lutionary terms, it seems that adult woundsmay be optimised for speed of healing and thismay result in an excessive TGFP, release. Inresponse to tissue damage TGF3,B is releasedby platelets. TGF31 induces local cells to pro-duce extracellular matrix and more TGFP,. Apotential regenerative response may be over-run by a cytokine surplus leading to scar for-mation.2 5 8

Evidence of a failure in the regulation ofTGFP1 has been implicated in experimentalglomerulonephritis and disorders associatedwith interstitial fibrosis.3 5 Systemic applica-tion of TGFP, NAs prevented autoinductionof TGFI-mRNA, and limited the inflamma-tory response in experimental glomeru-lonephritis, and decreased the deposition ofextracellular matrix.3 In contrast with adultwounds with an excess of cytokines, cytokineprofiles in fetal wounds are reduced.' Here weshow that lowering the growth factor concen-trations of TGFP3 in a chronic gastric ulcermodel leads to faster healing with considerablydiminished scarring and infiltration ofthe ulcerbase with fewer macrophages and granulo-cytes.

In contrast, local application of TGFP,resulted in massive scar formation. Recently,Mustoe et al showed that treatment of partialthickness gastric serosal incisions with a singledose of TGF3,I accelerated the healing of thewounds.'6 In our experiments, application ofTGFI,B accelerated the overall healing ratecompared with the control groups.

This finding may be explained by thehypothesis that during the process of evolutionadult wounds may have been optimised forspeed of healing under adverse conditions.This may have resulted in excessive inflamma-tory infiltrates and an excess of cytokinesduring wound healing in the adults.2 Shah et almimicked this fetal wound situation in thehealing adult rat skin wound by local injectionof an anti-TGFl3 NA to reduce TGFI31concentrations. This resulted in diminishedscarring in adult wounds and almost normaldermal architecture. Injection of TGF,81 alonehad the opposite effect.6

Macrophages and activated platelets are theprincipal source ofTGFI3 in the adult wound.

In the foetus, wounds heal with very littleinflammation, and the reduced concentrationof TGFP1 may reflect the absence or minimalmacrophage infiltrates in those wounds.2 5Our study showed a reduction in the

number of macrophages in wounds treatedwith NAs. But in wounds treated with TGFI31there was no significant difference in thenumber of macrophages compared withcontrols.For ulcer relapse, excessive deposition of

scarring tissue has been implicated as a riskfactor.4 Here we show that lowering the activegrowth factor concentration of TGF31 bylocal application ofNA at the ulcer site accel-erates ulcer healing and improves the arrange-ment of the tissue architecture of the healingulcer.Our findings suggest that an anti-TGFPI

therapy modality in gastric ulcer disease maybe a novel means of stimulating and improvingulcer healing.A preliminary report on this work was presented in abstractform at the Digestive Disease Week, San Diego, 14-17 May1995.This work was supported by a grant from the J and F

Marohn-Stiftung.

1 Whitby DJ, Ferguson MWJ. Immunohistochemical localisa-tion of growth factors in fetal wound healing. Dev Biol1991; 147: 207-15.

2 Adzick NS, Lorenz HP. Cells, matrix, growth factors, andthe surgeon. The biology of scarless fetal wound repair.Ann Surg 1994; 220: 10-8.

3 Border WA, Okuda S, Languino LR, Sporn MB, RuoslahtiE. Suppression of experimental glomerulonephritis byantiserum against transforming growth factor P 1. Nature1990; 346: 371-4.

4 Ishimori A, Kawakami K, Inoue S, Kano A, Takahashi T,Asaki S, et al. Predictors of relapse in peptic ulcer.Hepatogastroenterology 1992; 39: 396-9.

5 Roberts AB, Sporn MB. The transforming growth factor-13s. In: Spom MB, Roberts AB, eds. Peptide growthfactors and their receptors I. Berlin: Springer Verlag,1990: 421-32.

6 Shah M, Foreman DM, Ferguson MWJ. Control of scarringin adult wounds by neutralising antibody to transforminggrowth factor P3. Lancet 1992; 339: 213-4.

7 Shiann P, Liao CH, Chen SH. Histological maturity ofhealed duodenal ulcers and ulcer recurrence after treat-ment with colloidal bismuth subcitrate or cimetidine.Gastroenterology 1991; 101: 1187-91.

8 Border WA, Brees D, Noble NA. Transforming growthfactor-beta and extracellular matrix deposition in thekidney. In: Koide H, Hayashi T, eds. Extracellular matrixin the kidney. Basel: Karger, 1994: 140-5.

9 Guglietta A, Hervada T, Nardi C, Lesch A. Effect ofplatelet-derived growth factor-BB on indomethacin-induced gastric lesions in rats. ScandJ3 Gastroenterol 1992;27: 673-6.

10 Konturek SJ, Brzozowski T, Majka J, Dembinski A,Slomiany A, Slomiany BL. Transforming growth factoralpha and epidermal growth factor in protection andhealing of gastric mucosal injury. Scand J Gastroenterol1992; 27: 649-55.

11 Konturek SJ, Dembinski A, Warzecha Z, Brzozowski T,Gregory H. Role of epidermal growth factor in healing ofchronic gastroduodenal ulcers in rats. Gastroenterology1988; 94: 1300-7.

12 Szabo S, Folkman J, Vattay P, Morales RE, Pinkus GS,Kato K. Accelerated healing of duodenal ulcers byoral administration of a mutein of basic fibroblastgrowth factor in rats. Gastroenterology 1994; 106:1106-11.

13 Kobayashi K, Tominaga K, Kim S, Fukuda T, Higuchi K,Nakamura H, et al. Expression of genes for transforminggrowth factor-,B1 and components of the extracellularmatrix during gastric ulcer healing in rats. Gastroenterology1994; 106: 108A.

14 Mustoe TA, Pierce GF, Thomson A, Gramates P, SpornMB, Duel TF. Accelerated healing of incisional woundinduced by transforming growth factor-,B. Science 1987;237: 1333-6.

15 Tarnawski A, Stachura J, Krause WJ, Douglass TG,Gergely H. Quality of gastric ulcer healing: a new, emerg-ing concept. J Clin Gastroenterol 1991; 13 (suppl 1):S42-7.

16 Mustoe TA, Landes A, Cromack DT. Differential accelera-tion of healing of surgical incisions in the rabbit gastro-intestinal tract by platelet-derived growth factor andtransforming growth factor, type ,3. Surgery 1990; 108:324-30.

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