Thrombospondin 4 expression in skin

ESDR I JSID I SID Abstracts

0766

0763 FEA’IURFS MODEL&m

OF KELOID TISSW CAN BE MIMICKED IN AN U-4 VITRO PLBROPL.&SlA L. Newstead. Samandm O_di John P. Huneins. Dads Greiliw pfizcr Ltd..

Discovery Biology, Tissue Repair, Ssndwich, Kent. U. K. K&id is a uniaue human dermal t%mnroliferati”e disorder and is chaacterised

THROMBOSPONDM 4 EXPRESSION M SKIN Laila Nemuz-Ot m; LnstihrteofBiochemis&y, University of Cologne,Cologoe,Germany

colbre.s. Fwhemwre,

Thrombospondin 4 is a member of the thmmbospondin (TSP) family of exb’ecellularcalciumbinding matrix proteins. To date five members of tbls family are koown- TSPl.TSP2, TSP3,TSP4 and TSWCOMP (cartilage ollgomcric matrix protein) On Lhe basis of their primary stmcfllres the rhrombospondins ceo be divided info two subgroups: TSPl and TSPZ are homotimeric proteins (subgroup a). whereas TSP3,TSH and COMP (subgroup b) are boolopentamenc pro&s. TSP 4 is a 600 kDa glycoprorein of five ,de”,,cal subomts coooected by d,s”i,ide liokagea. Each monomer contains a globular amiooteiminal hepain binding domain, e pememensiog domam, four EGF-like ‘ype 2 repeats, se”eo calcun bindmg type 3 repeatr and e globular cerbaylterminal domain. Only linle d&e exist aboo, the ditxlbotion and function of dus pmlein. TSP4 is koown 10 be present ponoetally a well es in embryos. The tmoserip, has been deta?ed immunologically in the developing and adult newoos system. Moreover, TSP4 mRNA has been detected in early osteoblests,especially in the adage of the bone collar of developing long banes. These data suggest a role for this protein in Ule initisl stages of osteogeoesis and newel development. In adol, tissue the mRNA of TSP4 is cooceoeated io the hea,, skeletal muscle and in bmm. In this study we wanted to investigate the expression of TSP4 in skin. 7herefore we amplified ,he full-,en@b eDNA of ref.TSP4 by RT-PCR. The PCR prcd”c, wes rubcloned into an c&ryo,ic expression vector and nasfec,ed into 293 cells. A polyclonal antibody wes prepared against the purified recombmao, pmtem. This polyclonal antibody will be used to io”es,ige,e the expressionpanem of TSP4 to oormel and wounded skin. To estimate the domain foodion relationship of TSP4 we expressedbuncated recmoblneots of the full-length TSP4

the arrangement

Binding studies indicate interaction of this protein with collagen.

bv an

excessiveaccumulation o~exbacelluler matrix, m&ly collagen. In accordance with already publkhed da, OUT hisUllogic.4 stodics of human keloid tissue using a “a&y of staining methods have demoos,m,ed chick bundles of collagen which appear chemcterisdy “glossy” sod are no, seen in normal skin or soar tioe. Another important ex,mcellular matrix protein, fibroxetin, was detected in similar emooots as in wrmal sWn. 7he number of fibxblans wes comparable u, normal skin or

of

slightly increased. Only a few these cells could be idenlifti es myoIibroblests by labeling with asmooth muscle satin. However, a significant accumulation of inflamma,ory ceils wes found in ,be lesions. Sane of thcsc features cwld be mimicked L” a fibmplasia model in vifm. Adult human 6nna.l fibmblarts from a keloid lesion we,e se&d Bt IW% coo,loeoce in 24well plates and cultured in serum-rich DMEM supplementedwi,b proline. escorb&e and TGF-P. For control purposes, normal adult human damal fibmbk,sts wac cultund under the same conditions. During a culture period of UD to 3 weeks. normal and keloid fibmblws snatiflcd and dmositi exhacellular metrix. mainl” &egm.

lo mamlayu on tissue cul,oe plestic keloid cells @es more slowly than normal fibrot&

Howevc,, in rcspoae (a TGF-$ ulcy pmlifem,ed in a simile, way IO normel adult human demnl fibroblasts over ,be period of 21 days. Stnxiflcetioo oceored a, an earlie, fime point lban in cootml

k&id tibmblasls pmduced mwe fihrillar collagen than the contml calls and of these Bbrs appeared abnamal. In some cullurs the collagen bundles

demanstnrtcd ule “glossy” maphalogy which wes ohsewed in keloid tissue. Io cooclusioo, some of the feaulres of keloid tissue can be mimicked in in “iwe keloid flbmbles, cultures. Further hvelopment of the in drro syswn may facililate ondenbmding of (he functional mechanisms involved in ulis disease.

0767

0764 COLLAGEN EFFECTS

I AND OF

COLLAGEN

TOPICAL

III EXPRESSION

RETINOL

IN

TREATMENT

YOUNG OF

AND 8Ot

OLD YEAR

SKIN:

OLD

lNDlVlDUAL.S. James Vamni. Roscoe Warner. Sewon Kane. Garv J. Fisher and John J. Voorhees, Depts. of Pathol. and Demxitol., The Univ. of Michigan, Ann Arbor, MI. Replicate 4-mm punch biopsies of sun-protected skin were obtained from a number of young adults (18-29 years old) and from 50 individuals, aged 80 years or older. following treatment for I days with 1% retinal under occlusion or with vehicle alone. Biopsies were used for i) histology/morphometry; ii) immunostaining with antibodies to the collagen I and collagen Ill amino-propeptides; iii) biosynthetic incorporation

of 14C-proline

into pepsin-resistant

material

or assessment

of collagen

I

(al) and collagen lIl (al) mRNA by RT-F’CR; and iv) isolation of dermal fibroblests. Results of these studies were as follows: Collagen I and collagen JIl expression (by immunostaining) was reduced in 80+ year old skin relative to skin from young adults Topical retinal treatment for I days increased the number of fibroblasts present in the old skin (p&.05): immune-propeptides;

increased hnmunostaining for both increased 14~.proline incorporation

collagen I and collagen ill into pepsin-resistant material

(~~0.05); and increased collagen I (al) and collagen III (al) mRNA expression (p&.05 for bath), relative to vehicle-treated skin. Fibroblasts grown from punch biopsies of X0+ year old skin demonstrated increased proliferation (p
0765

0768

COLLAOEN GENE EXPRESSlON PATTERNS BY NORMAL AND KELOID PIBROBLASTS IN < ‘W THE THREE-DIMENSIONAL CULTURE pSaro. Dq7armw”l of Dermatolugy, c,u,,ma “nlvendy Schml u‘Med,c,na. Maehash,. Ciunma, Japan Recendy, we have estlbhsbed B oo”e, fibmblast culture syram supplementedw,h L-ascorb~ aad 2-

CYTOKINES DIFFERENTIALLYMODULATE MATRIX-METALLOPROTEINASE EXPRESSION if&TH;EE-DIMEyS;;N,AL COLLAGEN MATRICES. &JIZ Rou Wona. Sy e ossodo. Mc e MO an and Irene F Koohev& Wellman Laboratories of Photomedicine, ‘Department of Dermatology, Massachusetts General Hospttal, Harvard Medical School, Boston, MA While dermal fibroblasts receive only a small fraction of the solar ultraviolet radiation (UVR) incident on the skin, they respond to cytokines produced by keratinocytes exposed to UVA. Cytokines may affect extracellular matrix turnover by modifying the balance between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). We have addressed the question of whether UV-induced cytokinss such as TNF-o, IL-lb and IL-10 modulate the expression of MMP-1, -2, -3 and -9 and TIMP-1 in human denal fibroblasts grown in monolayers and in free-floating 3dimensional (3D) collagen l/Ill gels, a model closer to skin. Basal production of all MMPs, but not TIMP-1, was significantly higher in 3D gels, as compared to monolayers. While TNF-a and IL-i@ induced modest to high increases in the levels of all MMPs, the response of fibroblasts in 3D gels was different, and overall less dramatic, to that of cells grown as monolayers. IL-10 only induced a 2 fold increase in the level of MMP-1 in monolayers, but had no effect on the other MMPs. TIMP-1 levels remained unchanged by all cytokine treatments. These data emphasize the important differences between traditional monolayer cultures and 3D gels, a more physiologically relevant model of skin, not only in their constitutive production of MMPs. but also in their responses to UV-induced cytokines.

phosphate (A*_ Z-P). to wb,cb damal ftbroblasu o,gan,ze il delm,s-bke ~h,,cw,e by ,b.z accomuinuon of self-pwdoced en,race,,u,ar mn,ox. The purpose ofrh,s srudy II to exilmme collagen gene expresslo” by “WOUS types of scar hbroblasts as we,, IS oo,ma, fibroblastr o, ,brs three-d,menrnooal cul,“te sys,em in comption wid, mooolayer cu,,u,~ system. We obtaued ,oounal ,ibroblsrts from skin of S normal ,ndi”,duals w,d, “ary,ng ages a, operanon. Keloid. hypertropb,c sew sod aoophtc scar ossue ~Pcc,me”swere obrained by skm b,opsy or srug,cvl remo”al af,er obtainrng M mformed coorent Cells were grown o, Dolbecco’s momfred Eagle’s medun supplemented wtb I”% telal cal, semm m the presenceor absence of I mM Asc 2-P Cells were harvestedfof RNA e~oacoon at md,cu,ed tones end noirbem blo, eoalyaeswere performed Jo rbe monolayer culture. rbere wus no stgnifumr diffeteoce in collagen ,nRNA exprewon amoog tibroblas~ oi dxfkm,t orwns. Normill bbrablarts kept hwber level of proal coUagen sod proal(lIl) collilgeo mRI\‘A expressionul the monolayel coltore. whclear rbesc mrssageer; were _wadduu,,y decrearcd I” the doer-dunenrtoe:il CU,,UC ‘The levels “f ‘olla&?r” “fiNA expresston by atropiuc sea fibroblasrs were ‘“mpilmble I” thora “f nolma, flbloblrru lli the tblecdimens,onal culmre. In contrast. the rnersages d,d no, decrease ,o keloxd and hypertrwhx s‘di f,brob,asrr ,n rbc three-d,“e,,stona, co,o,re When we colnpaled rbe mRNJA erprers~on betweell tibroblasw hum rbe ceotre all* edge of t,,r ,.S,OI~Eof bypcrtrophlc ICIU cells bOlli ,br ‘entle siwwcif a decreased level o, collagenuse mRNA cxpress,on The= aes”l,s sugges, thdt ,,bloblast, +,“lll kelo~la and hyperu-oph,c scars msy have e defecuve dow-regulanon sysreln regulared by exlracell”lill o,a,,~ce~ AS tb,$ novel three-d,me,,s,ona, ~nlrure system ciln provide e o,a>e phys,alagud culrurr condmon for frbroblaats. we coosade, d,harour c”,,“IB system ni,~br he a good rnperoneoral model to eluc,date rhe pa,hogener,r o, fibrooc dmxders