AD-A279 86511 1A D _ _ _ _ _ _ _ _

CONTRACT NO: DAMD17-84-C-4108

TITLE: STUDIES ON WOUND HEALING

PRINCIPAL INVESTIGATOR: Rajendra S. Bhatnagar, Ph.D.

CONTRACTING ORGANIZATION: University of CaliforniaDepartment of StomatologySan Francisco, California 94143-0650

REPORT DATE: April 1, 1992 D TICELECTE

TYPE OF REPORT: Final Report JUN 1994L J

PREPARED FOR: U.S. Army Medical Research, Development,Acquisition and Logistics Command (Provisional),Fort Detrick, Frederick, Maryland 21702-5012

DISTRIBUTION STATEMENT: Approved for public release;distribution unlimited

The views, opinions and/or findings contained in this report arethose of the author(s) and should not be construed as an officialDepartment of the Army position, policy or decision unless sodesignated by other documentation.

94-1623394 5 31 059

Form ApprovedREPORT DOCUMENTATION PAGE OMB No. 0704-oo U

Puiec reorting burde fo this tcKfI/Oct iOnf nfo,'maliOn t nt nlltedl tO avrerag I hour Dr reipofit. .nflIudifg th@ tuiie for revreiewg .lfIltrUCtiOfl. teetrchlig Cansting datal souraCeslgattierang ari matansanin the data needed., and cOmpletng and resviewin the cofleclson of information tend comments req•ardingl this burdlen estimate or any other asec of thiscollect•in of informaitio•, cluding suggestiOns for edI ng this burden to Washington Headquarters ervices. Otrctaorate for information O•at•ons Ind Repons. 115 JeffersonOsiis Htghway, Suit* 1204. A=lington. VA 22024302. and to the Office of Management and Budget. Paperwork Reduction Project (0704-0 416). Washingon,. DC 20$03.

"1. AGENCY USE ONLY (Leave bdank) 2. REPORT DATE 3. REPORT TYPE AND DATES COVERED

1 April 1992 Final Report (8o1184 - 10/3114. TITLE AND SUBTITLE S. FUNDING NUMBERS

Studies on Wound Healing Contract No.DAMD17-84-C-4108

"6. AUTHOR(S)

Rajendra S. Bhatnagar

"7. PERFORMING ORGANIZATION NAME(S) AND ADORESS(ES) 8. PERFORMING ORGANIZATION

University of California REPORT NUMBER

Department of StomatologySan Francisco, California 94143-0650

9. SPONSORING/ MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING/ MONITORING

U.S. Army Medical Research, Development, AGENCY REPORT NUMBER

Acquisition and Logistics Command (Provisional),Fort Detrick, Frederick, Maryland 21702-5012

"11. SUPPLEMENTARY NOTES

12a. DISTRIBUTION / AVAILABILITY STATEMENT 12b. DISTRIBUTION CODE

Approved for public release; distribution unlimited

13. ABSTRACT (Maximum 200 words)

The overall objective of this research is to examine the feasi-bility of accelerating wound healing by understanding the intra-cellularcellular mechanisms involved in the regulation of collagen synthesis.Collagen is a key component of all tissues. It is involved indefining the architecture and biomechanical characteristics of alltissue and organs and plays a major role in the modulation of cellproliferation, migration and differentiation. Collagen synthesis is acrucial step in wound healing.

14. SUBJECT TERMS IS. NUMBER OF PAGES

Collagen, Lab animals, Rabbits, RodentsRAD II 16. PRICE CODE

17. SECURITY CLASSIFICATION 18. SECUITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF ABSTRACT

OF REPORT OF THIS PAGE Of ABSTRACT

Unclassified Unclassified Unclassified UnlimitedNSN 7540-01-2B0-5S00 Standard Form 296 (Rev. 2-89)

Prescrind b ANSI Std. Z39-.8

Summary

The overall objective of this research is to examine the feasibility of

accelerating wound healing by understanding the intra-cellular

cellular mechanisms involved in the regulation of collagen synthesis.

Collagen is a key component of all tissues. It is involved in defining

the architecture and biomechanical characteristics of all tissue and

organs and plays a major role in the modulation of cell proliferation,

migration and differentiation. Collagen synthesis is a crucial step in

wound healing.

The present studies are based on our hypothesis that the synthesis of

collagen is regulated by the superoxide free radical (020). Superoxide

is produced in large amounts by phagocytes which infiltrate a wound

in the early stages.

In the first two years of our study, our objective was to demonstrate

a mechanistic relationship between (020) and collagen synthesis and

prepare the background for extending these studies to in vivo

animal models in the continuation of the project. An in vivo wound

chamber model using rabbits was adapted from our collaborative •or

studies with colleagues at UCSF and used for initial studies on wound Corr0

healing. As the Contract ended at the end of the initial two years, we io

have reportable data only for the effect of (020) on collagen synthesis

in the in vitro tissue culture model for wound healing. _

Avakilc1iity G•Is

Dilt Speclal

Our studies showed that IMR-90 fibroblasts derived from human

fetal lung tissue are a suitable model for investigations on collagen

synthesis. These cells retain many of the biochemical characteristics

of fibroblasts in tissues. We examined the synthesis of collagen and

the levels of the collagen related enzyme prolyl hydroxylase in these

cultures in the prtesence or absence of (020). Exposure to (02')

resulted in a marked stimulation of collagen synthesis and an

increase in the activity of prolyl hydroxylase. We also examined the

nature of collagen synthesized in the presence and absence of (020).

These experiments showed that (020) increased the synthesis of type

III collagen to a much greater extent than the syntliesis of type I

collagen. This observation is consistent with the biochemical events

occurring in repairing wounds in vivo. Thus our studies supported

our hypothesis that an increase in (020) levels increases the synthesis

of collagen and suggest that measures to increase (0Z) may also

promote collagen synthesis in healing tissues in vivo and facilitate

wound healing.

-2-

Introduction

The role of oxygen wound healing is well recognized and documented

in our original proposal. The availability of oxygen is critical for

repair processes. The beneficial effects of hyperbaric oxygen

therapy on wound repair have been amply demonstrated. Increased

oxygen supply promotes wound healing whereas decreased oxygen

supply retards repair. The beneficial efects of oxygen on wound

healing can be directly correlated with its direct involvement in

collagen synthesis which is crucial to repair. Collagen is responsible

not only for tissue architecture and biomechanical characteristics, but

also plays a regulatory role as it modulates cell proliferation,

migration and anchorage, and contributes to cell and tisue

differentiation. Oxygen is a substrate for the extremely important

post-translational hydroxylation of proline and lysine residues in

collagen.

Many lines of evidence suggest that oxygen may also act at the

genetic and epigenetic levels to modulate the expression of collagen

genes and the expresion of genes for the enzymatic machinery

required in the post-translational modification of collagen. The

active species in this regulatory role of oxygen is not the oxygen

molecule but a product of its metabolism, superoxide free radical

(020). Superoxide is the product of univalent reduction of oxygen, the

first step in the biological fixation of oxygen. The (020) free radical is

a highly reactive species which can react with a variety of important

molecules and macromolecules in the cell, however, it is well known

-3-

that its interaction with the genetic material of cells results in a trin

of events that results in the unmasking and increased expression of

certain genes. Our hypothesis is that the (020) free radical is a major

stimulant for collagen synthesis and that it should be posible to

improve the efficiency of wound repair by increasing the tissue

levels of (020) free radical. In fact during normal wound healing, an

important event is the generation of (020) free radical by phagocytic

cells of the immune response which sets the pace of repair. It is our

belief that an understanding of the regulation of collagen syntheis by

superoxide can lead to the development of procedures for promoting

wound healing, especially under those circumstances where oxygen

delivery may be impaired by hypervolemia and other trauma

related factors. In these studies we have examined the participation

of (02) free radical in collagen synthesis to confirm its involvement

in the regulation of collagen synthesis and to elucidate the

mechanisms involved in the modulation of collagen synthesis. Our

studies are summarized in the article reproduced in the following

pages.

-4-

Celiuerwep4.VkcadaRWOlgy 37(7). 751-755. 1991 0 145-5680&91 S3,O00 +O-Pianted in Great BDelta,. 0 1991 Pergamon Prtss PIC

STIMULATION OF COLLAGEN SYNTHESIS IN FIBROBLAST

CULTURES BY SUPEROXIDE

Gowu, CHANDRAKAS AN' AND RAJUNOA S. BHATNAGAR

I Cenral Lmasla Resmewc Insliujw Adyw. Wle* -E6W 020. 15db.Labacaiw of Connmetiv rmna Biochemamy. Boa 0650. C421W. School of Dentuivy,

Univaicy of Caiafornin. Sm, Frncuc. CA 9414340650. USA.

AccepeeS A*Swt 31. 1989

Abehact Exposmr of diploid total hau- rabroblass (IMR4O0) to mipmumios usmuini bydihykhoayfummmsrsle maw to auxnmd collaps s)Iusm. The s e of tmp MU coiqim wo

*uabok~doby saqasoujdidsdAmm. Oiobgsrmvuioe gmvjpw=Wmiry payamls morepdMomofcof qmsyi~mdasymouininymod~dfaubolpneape.5dOLThuchmvaftmay explala doe increasd Sisymhsof COlbsA a Mm asisllowing b5kfk oearspininooWW

Key ward Supseoxides. sltulaini. cobmlhm syinbeMls yeIcoilqm. tpecoiqoM.fbmsbb

STIMULATION DE LA SYNTHESE DU COLLAGEN! EN CULTURES DEFIBROBLASTES PAR LE SUPEROXYDE

Rdsom* L'ezpomskmodsfibmbobnimdphlndsm U h midn (1b134 Ads qul~ude gimbi paIs hdiby~oyumwm etmanhi- iagmmsaime db sywubhsdscolqlim.La I il decoblmeitype IIIaddsmal &muhshdsuophfotqwalledscoqllsmeypeL mdewdm~bhehdo

'as iftle d IabouIaadel ) wihedecollhg atueinmm oidmameI uinmddbinsbsbeeadmoolblin.Cesalb muan-ps Imaplm d~nnodby ee~colsondmka sn&su mam an wflaumio anl Am exapuuhiA don comilidon oxydue

INTROUcIIoN

Sevad U linesMvkdmu suggeu a tabefor oxygm wAd is meinholis in die mpulda of collGe synhesi&Inflinain, ap logcalcudiden diwuin~ad by dft So1I In of tupesuxadb &e =15Wem 03011

to exame collage sydIbesis amd depoedom mn namsii (Dunesf at .. 1976; Kmr. a a.. 1976) The syadisis ofcollage dwiumS -m o apsi is inwdukais by due sv~ailab y of omygma (Knigin a a.1. IM6). Expomu of

saussin soypsaoui condinm (awap ad wAPeng, 1975) ad odmt*M so*he amine (Dh@MaW~.. 196MesniW ia imumud colla syndmeis In odddm, ag. vach as Sharycia mid Peaque (12u91W vialog)

of dieupnn of inflaamndat in Alauds diusnee it is of isat, to eamuin die muhalmsý invold is*A dmusma collage syad=si under adwime ndidom. We haoe inveilged diesyndiedh Of coll am

die ~u ofc otzoeaus 03 i IM -W diplul fftablan.

-5-

752 Chandnakasan, G. and Bhamagr, R. S.

MATERIALS AND METHODS

CCUi CaiWe Prixedaes,DitAoad librobhsts (I)MR.90 derved from fttal huma lung were obtained in the rafth and sixth pas" from the Instiute forMedical Rteseueh.Camade. NJ. Al leapeimeasswere caroidou ia n tim esiblao twelfth puNSSS. The cells wen2 Xmwn toconfluencein 60 m dishes (Falcon Plastics) in Dulhecco's moddifcation of Eagles minimum essential medium sigopiemented with i0% ftalbovineseu.

Eapourwi of Fihroblu s wSmperau&dTheceUswmwesposedwoo 1 indw th ,rm otldihydroxylumarm (DHF). a well leaow genermoriofthe free radical ihe.91)Prveious smiths (Autor etat.*. 1960) showed Mtha 011 genaerawsO 1 0.n tamue culture media and that the free radical produced underti'*se conditions is scavenged by msupeoxide dismaimase. We used this agent because its ouher metabolites ame not known to hsewsieaafkait effects o. cels. sad therefoe the observed WeNWu effects can be auributed to the protuctiom of oxygen metaboigneisthe Culture mediumsPreliminary experwaseas usin Trypen Blue exclusac wsay showed no cymtoxc effects us the presnce of DIIF ittcncevmused ndestexeriineni. The tkofasignifcuwcytotoxicity a DHooncaniasmolowerdtd0oOIM wascoakmnebyexpegmimenmin which the incorporation of radiolahelled precursors tom cei llui macomolecules was examined.

Coilqes BlosynrhwskCulture jeosms were lsaieled with tOX ~Ci U'4C1 proline (268 mCahibnoe. New England Nauclea) for 24 lvi. is the I ecof3-mpoioml~ ino mbisorofcoilagecosliniuing(Pucor mad Tud.. 1982) to acilivistaw U.mmysutcollsse. Thiagent is not kaminwafmaet Oiot SOD. For analysis of radiosctive incorporation and Collagen syntheis the metham and ntll b~wwene harvued maddialyzedasputa H0i thep esee ,of ImMMehylenediafnetmm aaetic acd ad I Mphemylmogmitykslonyfluorde a 40. Contro expenimems showed over 95% recovery of labelled! poumsi by this p 9 Pth1e1 Th dialyzed samples werhydkolyaed a 6 N HC1 for 24 lmat. 110 land the hydiflyzall ware mastlyzed for ( 4C)-hydrosypolise by published mesbods (JimvaaidPvockop. 1966). DNA conmnIasamesmae of cell ponlt wadeuurmined by thedipisylassia method (Bame.. 1956) Toldprames eoma -was derminid by Coouassie Br~isi Blue procedure (Dradkat 1976).

Andlysis for Type I &%d Type /it Co~algenIn order to iWOWa and quwmasem the newly syndiesiand type I and type III collaseas. both the condkioned methan 410d 11111 ad I@Y

were subjecs to limied papsin digesuuon to acilkmae hecompilete s dolbina.sd chein sepainsonotthecollhefs. Thecollagewee sepamud by fractional sak precapdataon with 1.1 M and2.6 M Nsa atpH 7.0(Mfllerandl Rhoedis. 1962) The labelled eotie.clminwee Iep- "by chromaoga~by an column' of caboxymethyt colde.. The ideaitey adof echains wee camfomned by

plyaoshids sa elecuophom

RESULTS

In preliminary experiments (data not shown), we detainwned the optimal concentraion of DHF for use in thenestudies. Since hydroxyproline synthesis occurs as a result of intracellular hydroxylation of proline incorporatedin polypeptide precursors of collagen. we followed the synthesisof iCIhydroxyproline asa measure of collagensynthesis The amount of radioactivity incorpome dinto hydroxypioline measured as a function of DNA contentincreased in t ,he presence of DHP. Maximal increase in hydroxyproline synthesis was achieved at DHFconcenrataons of 10-20;&M and no funher incemai occurre beyond these concenvations. Alt subsequentexperimentswere carried out atDHconcenwationsof 1I0LM. Maximal collagen synthesis in fibroblasitculturesoccurs when the culturesibecome- confluent (ftulaf~sky. 1972). All experiments inpone here were carriedout with confloencultuuus. In onler sexamune the possibility thai the effect of DHFon collagen synthesis mayhave resultd hum am increase in the nwnbier ofacels we deuuuIdnud the weAl DNA and protein conten of thecultures (Fig. 1). Eaposmuofcells to Oforperiods Wpo 24 hma did not resultin any increases in DNA orproeeicommnt indicatial; th the fuee radical did not induce cell prlifuration in the coinfluent cultures. Mhe increasein I'M1 hydroxtypriline synthesis as a hundon of the DNA content of the cultures was appmarn as early as 6hms afte the a~ddton of DMF (Figl. 2). The increase was linea during the 24 hrs period of observaumoe

in order toconfimn if the incemase in collagen synthesis in the presence ofDHFIresulted from ge -nmaom f02the effectofremoving the fre radical by suproxide dismucse was exatrined. As see in Ta"l I. expoesmeotculturles to 10 pM DIIF for 24 In. seulted in a 23% increas in the incorporation of ["QC1prfmuln ee a 130

Stimulation of collagen synthesis by superoxide -53

-1 - - - -

Fig. I Fig. 2Fig. I TDo lack of effect of DIIF on cell growth: Confluent cultures wer exposd to t0 pM DH]Ffor 24 hrs. and DNA and poven content were deteramned as descnbed. Each vaue is the mew aotriplicaic culture. Open crcles and mangles represent data from contrl culagec r'led cclus

and mangles replesnt DHF-ureated cultures.

Fig. 2 Increased collagen syntesis in the presence of DHF: Confluent ciusns were exposed to10 u.M DHF and labelled with ("Cf-prolmne for vwno• tkme. The content oqf]I-hydoxapdlmi

was dearmined as descnbed in Methods. Each point is the avears oaf tpira culnaes. Closedcurcles represent conl, and open circles represent dAt from DF-HPmved culuss.

TABLE I 114 CI . Proline Incorporaon and Synthesis of 114 C1-hytrocyprolim in the presence of DHF

Radioacuvity incorporated (dpm4Lg DNA x 104)

Treament Total Radioacuvity . 14 CI-bhydrxyproline

Experiment I Conmol 188.7 ± 21 3.39 t 0.33+ DHF. 10 Aiml 231.5 ± 22 (+23%)" 7.81 t 0.77 (+130%)06+ DHF, 10 gs/ml + SOD 190.0 t 18 3.83 t 0.39

Experiment 2 Contol 159.6 t 19 2.76 t 0.30" DHF, 10 Pslad 211.9 ± 23 (+32%)* 8.03 ± 0.85 (+196%)**"u DHF, 10;L&/mul+SOD 161.2 ±"17 2.81 t 0.30

Corflum cutiuam wes mwud for 24 hn. in the premee of DHP and rCl)-lpm with ofwithos SOD. It ePWemWmuM with SOD. the Cal we qwlibwued in SODacmwning medium for30.. OwWNft a•i•dmsa of "IU. Is nobs ma uadteses adwase pae change tomr

cmoL ELat mWAmbr is the mm± S.D. of Omv deumawnumm * P 4 0.05, "" P 0.001.

increase in dh synthesis of ('C1-hydroxypolvine, on a per DNA besis. Since hydmxyproline is poeseft insiVpfcu amounts only in collagen. these damn sugea that the incmased incoporation of proline could beaccoumd for largely by increased collpen syndteis This nm was abise by supamids diumaeadded at acocetation of 10 U/nm in dew culture medium, Heat denratud superoxidedismah had nod faThese dama suppom dte concept thah d• mulamey effec of DHF o0 collagen syntwhss may be wedaW by 02

The enremso of collagen phenotypes is alimd in disues under condions characumted by ina:elasedpman002(Baileyetoa.. 197T Baumamnai.. 195L;ClaveetaL.. 19"9).W¢ezawnedd natmofcdllasytwu iid

"---• " -7--

754 Chandrakasan. G. ant! Bhatnagar. R. S.

in the presence of DHF. The labelled procollagens were subjected to limited pepsin digestion to ensure completerecovery from the matrix, and were differentially precipitated with NaCl to separate type I and type III chains.The chains were further purified by chromatography on carboxymethyl cellulose columns and the radioactivity)incorporated in type I and type 1H chains was measured. These data are presented in Table 2. As seen in Table2. t;. synthesis of type III collagen was increased by 42% whereas theme was no significant increase in typecollagen synthesis.

TABLE 2 Relative Synthesis of Type I and Type /I/ Collagens in the Presence of DHF

Treatment *Radioactivity, dpm x 10-4 Type Iltrype I

Type[I Type III

control 1.82 ± 2.3 0.32 ±0.35 0,176+DHF. lO0ig/ml 1.91 t 2.1 0.46 ±0.41 (+42%)** 0.238

ORadkdviiuy in pooled ftactions froin carboxymnediyl cetiulam chromatography of differenial ukprecipitawi of type I and type IIl collagens. Exh value is doe averagle of miplicu experiments t S.D.

T`he numnber in puendiesis denons percen changs tiramn control. 00 P < 0.05.

DISCUSSION

Our studies suggest that 0, may directly stimulate fth synthesis of collagen. Other studies in our laboratoryshowed that the free radical induces increased prolyl hydroxylase activity in IMR.90 fibroblasts. The increasein prolyl hydroxylase activity in the presence of 02 was ablse by savengersofthe free radicals. These damnprovide an explanation for the increase in collagen synthesis observed in tissue under conditions that elevate02 levels, including intlarumation (Babior, 1982), exposure to increased oxygen (Chvapil and Pent. 1975).oxidants such as ozone (B1hatnagar et at., 1983), and exposur to 0, generating agents such as paraqlust anBleomycin (Hussain .t al., 1985; Hussain and Shatnagar. 1979). The observation that anti-inflaimnuay agentsdecrease fth production of 0. (Oyangui. 1976) arid cause a lowenng of collagen synthesis (Kruser ata.. 1978;Phan er at., 19981)ilWso supports this concept.

Type III collagen synthesis is markedly enhanced in tissues in early stages of fibrosis and other =scaringprocesses (Bateman ei aL., 198 1; Kent et at., 1976; Kruse et at., 1978. Phan etat.. 198 1). Type III collagen alsoaccumulates in repair tissue during wound healing (Bailey etat.. 1972; Clove utrat., 1979). The greater increaseiin type flI collagen synthesis in the presence of 0, observed by us suggests that the free radical may play astolin the modulation of differential collagen gene expression.

The present studies do not provide information as to the mechanisms of regulation of coltagen synthesis by OrThe free radical is known to affect cell growth and differentiation (Michelson. 1982; Michelson andBuckingham. 1974). The genetic level regulatory effects of the free radical may arise from its interactio withinfaonstional macromnolecules ( Brown and Fhdovich. 19831; Leska er at., 190, Moody and Hassan, 19182).frnceracton of the hae radica with specific sites an the chromastin may result in the expression of the genieticcompisa ftr colilage synthesis, includng the enzymes involved in its .poustntnslational Processing.In highe organisms the inflammatory response elicited by foci of irritation such as injury, bacteria. orparticulate material elicits the synthesis of collage to wall off the mirriant stimulus. Collagen plays a protectiverole by forming a tough and impgrevous barrier against injurious stimuli including radiation and oxidans. Ithas been suggested that (Cemand. 1985) tissue pmooxidant state characterized by increased levels of oxygen-derived free radicals may induce the expressio of certain gemes that are a paut of the ogn~ism's defentsiverespnse. reminiscent of the SOS funactionl in bacteria. It is interesting to speculate that teleologically collageand its biosynthetic machinery may be a part of such pro-oxiduu mechanisms in animal cells

Stiniuano of collage syndbcss by superoxide75

Ack. danevie0. Cb&Waaa aKnowledp nPAon fom the Fulbrigha Ploprm Theme amadia Wa suwpopa by Mmi

frma ab Niation hud of Heaja ms, Selia Emmpqm prwvided gxpo wciinuu assisc

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IfidbsI m 4 AX. ad 3kcngiMw IS. Sfbm. af upseomid raftialsam mayobl growib aid differmaaduam Sieek.. Mh)nAmRex Com. 19M4. it 1079.1016.

Blip' e AM, Cann. mAaca Atom ai .eACm SuppL 198Z 11: 179-301.Nid.CS. dHM1 W KM..-- ldugmcayof oxygekn a6.. cab. Prc. mAea~ S.dUSA. 196279: !1265.239

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1962 62 305-319.

Conclusions

Our studies support the hypothesis that superoxide is a stimulant for

collagen synthesis. It promotes the overall synthesis of collagen, but

the increase in type III collagen synthesis is much greater than that

observed in type I collagen synthesis. Type III collagen is a fetal

type collagen that is involved in development and differentiation of

tissues. Increasing levels of type III collagen in wound tissues can

be expected to have beneficial effects on tisue repair and

regeneration. Our studies support the concept that increased

delivery of superoxide to wounds may accelerate and facilitate

repair. We suggest continuing studies along these lines to

* Examine the effect of superoxide on collagen synthesis in

vivo,

* Examine the effect of increased superoxide levels on wound

healing, and

* Develop strategies to administer superoxide to wounds.