characteristics of growth, morphology, contractility, and protein expression of fibroblasts derived...

Characteristics of growth, morphology, contractility, andprotein expression of fibroblasts derived from keloid

HAJIME SATO, PhDa, AR19AW1 SUZUKI, BSO,*, MAKOTO FUNAHASHI, MSa; TOSHIAKI TAKEZAWA, PhDo , *;YUTAKA 0GAWA, MDT; KATSUTOSHI YOSHIZATO, PhD-

Phenotypic alterations of kelold-derived fibroblasts were characterized by comparison with the phenotypes ofnormal fibroblasts from the same patient . Explant cultures of keloids showed unique features . Kelold explantscontracted considerably and reduced their size during culture, whereas the size of normal skin explants remainedunchanged . Enlarged cells were found among fibroblasts which had grown out of all the explants and weremorphologically distinct from fibroblasts ; however, keloid explants produced many more of them than did thenormal tissues . The growth rate of fibroblast colonies formed from normal explants was five times higher than keloidexplants . Keloid fibroblasts which had been serially cultivated contracted lattices of collagen gels at a rate similarto normal fibroblasts . Proteins extracted from serially cultivated fibroblasts were mapped on polyacrylamide two-dimensional electrophoretic gels . No significant qualitative alterations in protein expression in keloid cells werefound as compared with normal fibroblasts . However, some quantitative changes were found between the two . A

ter-assisted image analyzer detected 151 polypeptide spots-50 spots (33%) of which increased theiramounts in kelold cells, whereas 34 spots (22,5%) decreased in comparison with normal fibroblasts . Sixteen majorpolypepticles were identified as known proteins with the aid of time-of-flight mass spectrometry . The level ofexpression of these identified proteins was similar between normal and keloid cells, except stathmin whoseexpression was suppressed in keloid fibroblasts . (WOUND REP REG 1996;4:103-14)

The keloid is a sequela of injury to the skin of humanbeings and is characterized by the conspicuous growthof dermis both within the original wounded site andbeyond the confines of injury .' The mechanism un-derlying the pathogenesis ofMoid is largely unknown.

From the Yoshizato MorphoMatrix Project, ERATO,JRnC, 11 and Developmental Biology Labora-tory, Faculty of Science, Hiroshima UniversitycHiroshima,, Konsai Medical University Hospital,Osaka," Japan .

*Present address: (A . S) Institute of Immunology Co., Ltd,Tokyo, (T. T.) Japan Technical Center, Proctorand gamble Far East, Inc., Hyogo, Japan .

Reprint requests : Kotsutoshi Yoshizato, PhD, YoshizatoMorphoMotrix Project, ERATO, JRDC, 13-26,Kagamiyorno 3-Chome . Higashihiroshimo, Hiro-shima 739, Japan .

Copyright 0 7996 by The Wound Healing Society.1067-1927/96

55.00 + 0 3611171278

Yoshizato MorphoMatrix Symposium

PVDFSDSTOF-MS2D-

o's modified Eagle's mediumis focusi

Polyvinylidene difluorideSodium dodecyl sulfateTime-of-flight mass spectrometerTwo-dimensional polyacrylamide gelelectrophoresis

are involved inthe origin of keloids.2- "1 Keloid fibroblasts proliferateabnormally and produce extracellular matrixes ofwhich collagens are major components . Therefore, itis important to know the phenotypic characofkeloid fibroblasts whichdistinguish them from thoseof normal fibroblasts to develop a better understand-

mechanism of keloid formation .For this purpose, fibroblasts cultured in vitro ap-

pear to be useful resources for experimental work and

1103

104

SATO ET AL .

have been used to understand the characteristics ofkeloids at both the cellular and molecular levels .s How-ever, there may be pitfalls in studies using culturedfibroblasts obtained from these non-congenital dis-

eased tissues because it has been shown that fibro-

blasts in vitro are apt to lose lesion-specific pheno-

types expressed in vivo during culturing. Conversely,the explant cultures appear to be an in vitro modelwhich approximates in vivo characteristics.

The present study involved three experiments tobiologically and biochemically describe characteristics

of keloid-derived fibroblasts by comparison with nor

mal fibroblasts . Specifically, these studies examined

the following: (1) the growth, morphology, and con-tractility of fibroblasts in the explant cultures ; (2) the

ability of serially propagated fibroblasts to contract

collagen gels ; (3) protein expression of serially propa-

gated fibroblasts shown by two-dimensional electro-phoresis on polyacrylamide gels (2D-PAGE).

We foundthat in explant cultures keloid fibroblastsshow characteristics of growth, morphology, and con-tractility which differ from those of normal fibroblasts .However, similar phenotypic expression of protein and

collagen gel contracting ability were observed for both

keloid and normal fibroblasts which had been seriallycultivated .

MATERIALS AND METHODS

Culture of explants and fibroblastsTwo sets of biopsies of both keloid and normal skinwere obtained fresh and sterile from two consenting

patients whohadplastic surgery procedures performed

at Kansai Medical University Hospital . One set ofsamples was from the left shoulder of a 18-year-oldfemale patient (No . 1) and the otherwasfrom thebackof a 27-year-old female patient (No . 2) . These keloidswere diagnosed as "true keloid" and not as hyper-

trophic scar because the tissues had grown beyondthe confines of an original wounded site . Three andthirteen years, respectively, had passed at the time ofoperation since the initiation of keloid formation inthe two patients . Contractures were not observed foreither keloid. The biopsy samples were minced intosmall pieces 0.5 to 1 mm' with a sharp scalpel bladeand cultured at 37° C in 100 mm culture dishes (FAL-CON, Nippon Becton Dickinson, Tokyo, Japan) in anatmosphere of 95% air and 5% CO, with the use ofDulbecco's modified Eagle's medium (DMEM; GIBCOBRL, Grand Island, N.Y.) supplemented with 10% fe-tal bovine serum (Hyclone Laboratories, Inc., Logan,

WOUND REPAIR AND REGENERATIONJANUARY-MARCH 1996

Utah), 100 pg/ml streptomycin (GIBCO), and 100 U/ml penicillin (GIBCO). When fibroblasts which hadgrown out of the explants reached confluence, theywere passaged by treatment with 0.05% trypsin andethylenediamine tetraacetic acid 0 .53 mmol/L in cal-cium- and magnesium-free Hank's balanced salt solu-tion and subjected to serial cultivation as describedpreviously ." Medium was replaced three times a week .The growth of colonies was monitored by taking pho-tomicrographs at days 1, 11, 18, and 25 through aphase contrast microscope .

lmmunocytochemistry of fibroblastsEnlarged cells were found among fibroblast coloniesas detailed in the text. The presence of a-smoothmuscle actin in these cells was determined by im-munocytochemistry . Cells cultured for 35 days in35 mm culture dishes (FALCON) were fixed withcold ethanol and incubated with mouse monoclonalantibodies against a-smooth muscle actin (SigmaChemical Co ., St . Louis, Mo.) . Bound antibodies werevisualized with peroxidase-labeled second antibod-ies (Vector Laboratories, Inc., Burlingame, Calif)with the use of 3,3'-diaminobenzidine as substrate.To ensure the reactivity of the enlarged cells to theantibody, we overexposed cells to the second anti-body (over night) . Cells were counterstained withhematoxylin .

Collagen gel contractionFibroblasts at the eighth passage were embedded inthree-dimensional lattices of hydrated collagen gelsaccording to Asaga et al .ll The following stock solu-tions were prepared and kept at 4° C : 0.5% (w/v) col-lagen solution (bovine type I atelocollagen, Koken,Tokyo, Japan) : 5 x concentrated DMEM containingHEPES (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]) 80 mmol/L, NaHC03 40 mmol/L, strep-tomycin 0.4 mg/ml, and penicillin 400U/ml . Cells wereharvested from monolayer cultures as described pre-viously, counted, adjusted to desired cell number, andcollected by centrifugation in a plastic tube. The cellpellet containing 105 cells was resuspended at 4° C in2 ml of DMEM containing 0.24% collagen, HEPES 20mmol/L, streptomycin 100 pg/ml, penicillin 100 U/ml,and 10% fetal bovine serum. This solution had beenprepared by quickly mixing the stock solutions, fetalbovine serum, and redistilled water. Two milliliters of

the cell suspension was distributed to hydrophobic 35

mm dishes (FALCON) . The dishes were transferredto an incubator kept at 37° C and maintained in anatmosphere of 5% C02 and 02 95% air. Collagen had

WOUND REPAIR AND REGENERATIONVOL. 4, NO. 1

Figure 1 Gross appearance of explant cultures . Biopsy specimens of keloid and normal skin (patient No . 1) werecut into small pieces (0 .5 to 1 mm2 in area) and cultured . The growth of fibroblast colonies which had grown outfrom the explants was monitored for up to 25 days by taking photomicrographs through a phase contrast micro-scope . To cover an entire field of fibroblast colonies, photographs were taken for various regions of the opticalfield and assembled into a panorama . Explant cultures were obtained for patient No . 2, and identical resultswere obtained . Scale bar= 500 gym,

gelled within 10 minutes and cells were embedded inthe gel. One milliliter of culture medium was placedonto the gels, and medium was replaced thereafterthree times a week during the course of culture .

Two-dimensional electrophoresisof cellular proteinsFor each electrophoresis separation, two identical gelswere run: one for electroblotting and the other for theprotein staining as described later. Samples for elec-trophoresis were prepared according to Koseki andYoshizato . 12 Fibroblasts were lysed in 1 ml of a solu-tion containing urea 9.8 mol/L, 2% Nonidet P-40, 2%ampholyte pH3 to 10, and dithiothreitol 100 mmol/L(Millipore, Bedford, Mass.) . Samples were stored at -80° C after protein concentrations were determinedby the Bradford dye-binding procedure . Preparativetwo-dimensional polyacrylamide gel electrophoresis

SATO ET AL.

IOS

(2D-PAGE) was performed with the use of a MilliporeInvestigator System (Millipore). Proteins ofcell lysateswere separated by 2D-PAGE described by O'Farrell13

with some modifications . The first dimensional sepa-ration was carried out by isoelectric focusing (IEF) in3 .2% acrylamide tube gels (210 x 3 mm). The gels weretransferred into a tube containing sample buffer (10%glycerol, 1% acrylamide, 5% 2-mercaptoe thanol, Tris-HCl 0.2 mol/L, and 0.1% bromophenol blue, pH 6.8) .The second dimensional separation was performed inthe presence of 0.1% sodium dodecyl sulfate (SDS) ongels of 12% polyacrylamide (230 x 200 x 1 .5 mm) with4 cm high 6% acrylamide stacking gels andterminatedwhen the dye reached the bottom edge of the gel.

ElectroblottingAfter electrophoresis in the second dimension, thegels were equilibrated for 5 minutes with cyclohexyl-

106

SATO ET AL,

Figure 2 Contraction of kelold and normal skin expiants . To quantitatively compare the rate of contraction ofexpiants between normal and kelold specimens, the size ofeach expiant was determined on photomicrographsas shown in Figure 1 . Measurement was done for two sets of biopsy, Square, Normal tissue from patient No, 1 ;diamond, normal tissue from patient No, 2, circle, keloid from patient No . 1 ; triangle, keloid from patient No . 2.

aminopropanesulfonic acid buffer 10 mmol/L, pH 11.0(Dojindo, Kumaimoto, Japan), containing 20% metha-nol . The gels were then electroblotted onto polyvinyl-idene difluoride (PVDF) membranes (FluoroTransmembrane, Pall, Havant, United Kingdom) for I hourat a 250 m.A constant current in a Bio-Rod Trans-Blot transfer cell (Bio-Rod Laboratories Inc., Rich-mond, Calif.) . Proteins were visualized by stainingwith 0.1% Coomassie blue R-250 in 40% methanoland 0.1% acetic acid n 1 minute and destaVed for 2to 3 minutes in solution of 10% acetic acid and 50%methanol, The membranes were thoroughly washedwith water, dried, and stored at -20' C . These blot-

s were used for peptide fingerprintingas described later .

Image analysis of proteins ontwo-dihiensional gelsProteins separated by 2D-PAGE, transferrand stained were then scanned by a Bio Image Electro-phoresis Analyzer (model 60 S-21) ; Millipore) and con-verted to video images (polypeptide maps) . The opticaldensity of a spot was obtained by integrating all pixelsin the spot . This analyzer can compare the pattern ofdistribution of protein spots in two images which wereobtained from fibroblasts of normal skin and keloidderived from the same patient. The two images weresubject to a geographic correction and compared witheach other!2 Red spots in the present study representpolypeptides expressed only in keloid fibroblasts and


green spots polypeptide present ofibroblasts . Matched spots were shown in blue and rep-resent proteins common to in both images . Matchedspots were quantitatively compared between normaland keloid fibroblasts . When a difference in the inten-sity of a protein spot between the two was more than20%, this difference was judged as significant.

Peptide fingerprintingSamples far peptide fingerprinting were prepared ac-cording to Henzel et a1 . 14 and Sutton et al.,' ,' with slightmodifications as follows . Single protein spots whichhad been blotted onto PVDF membranes were excised,placed in 1 .5 ml microtubes, and wetted with I plmethanol . The blots were reduced for I hour at 45' Cwith dithiothreitol 7 mmol/L dissolved in 10011.1 ofTris-HC1 0 .5 mol/L (pH 8 .5), -10% acetonitrile, andethylenediamine tetraacetic acid 5 mmoVL. They werethen cooled to room temperature, and 1 ml ofiodoaceticacid 200 mmol/L and NaOH 0.5 mol/L was added tothe microtubes for alkylation, which was allowed tocontinue in the dark for 20 minutes at room tempera-ture. The blots were immediately rinsed with waterand then incubated with 200 ~d of 0.25% polyvinyl-pyrrolidone 40 (Sigma) in acetic acid 0.5 mol/L on ashaker at room temperature for 20 minutes to pre-vent protease adsorption to PVDF membranes. Re-sidual polyvinylpyrrolidone was removed by rinsingthe blots with water and then with 20% acetonitrile,Blotted proteins were digested with 0 .2 Vg trypsin at

WOUND REPAIR AND REGENERATIONVOL. 4, NO . I

Figure 3 Growth of fibroblast colonies in the explant culture . To quantitatively compare the rate of expansion ofcell colonies from explants between normal and keloid specimens, the area of each cell colony was determinedon photomicrographs as shown in Figure 1 . Measurement was done for two sets of biopsy . For explanation ofsymbols, see Figure 2 .

37° C for 17 hours in 50 p1 of ammonium bicarbonate0 .1 mol/L and 10% acetonitrile . Digested samples wereobtained by removing the pieces of blottedmembranesand drying . Dried tryptic digests were dissolved in 1

p1 of30% acetonitrile and 0.1% trifluoroacetic acid andmixed with 1 p1 of a saturated solution of a-cyano-4-hydroxycinnamic acid in 0.5 ml microtubes (Sigma).They were placed on a sample probe tip. Mass spectrawere obtained with a time-of-flight mass spectrometer(TOF-MS; REFLEX, Bruker, Bremen, Germany) .

Inputs for the analysis consisted of a list of tryptic

digested peptide masses, accuracy 0.1% and a protein

molecularweight range whichwas estimatedfrom two

dimensional electrophoresis. The number of allowedmismatches wasone. Theprogram scans the database,generatesaminoacid sequences of fragmentspredicted

by tryptic digestion, and computes the molecularmasses of the fragments. Spots were identified as

known proteins when the parameters were matchedto those of proteins included in the database. The pro-tein database used by the program was Swiss-Prot(Release 26, July 1993 ; European Molecular Biology

Laboratory, Heidelberg, Germany), which contains

over 31,000 sequences.

RESULTS

Explant culturesExplant cultures were obtained for normal and keloid

tissues from the same patient. Fibroblasts started to

SATO ET AL.

107

Figure 4 Phase contrast photomicrograph of cellular growth froman explant culture showing the presence of enlarged cells in thefibroblast colonies. A region similar to that shown in Figure 1 wasviewed at a higher magnification to show the enlarged cells . Pho-tographs were taken at day 25 of a keloid tissue explant culture .Enlarged cells also appeared in the cell colonies formed by nor-mal skin explants . However, the rate of their appearance in thesecell colonies was much greater in keloid-derived cultures than innormal cultures . Scale bar= 100 pm .

grow from the tissue after 8 to 11 days of culture. Asthe culture period increased, the keloid tissue con-tracted and reduced its size to about one third of theoriginal size at day 25, whereas the normal tissue didnot contract (Figure 1) .

The changes in explant size were quantified bymeasuring the area of explants periodically duringculture up to 25 days (Figure 2) . Normal skin explantsexpanded about 1.5 times original size during the first


Figure 5 Immunohistochemical detection of a-smooth muscle actin in enlarged cells . Cell colo-nies at day 35 were subjected to immunocy-tochemistry with a mouse monoclonal antibodyagainst (x-smooth muscle actin . A, Low magnifi-cation view ofa culture of normal skin explant cul-ture ; scale bar= 1 mm. B, The region marked by arectangle in A was observed with higher magnifi-cation ; scale bar= 100 pm .

WOUND REPAIR AND REGENERATIONVOL . 4, NO. 1

Figure 5 cont'd Immunohistochemical detection ofu,smooth muscle actin in enlarged cells . Cell coloniesat day 35 were subjected to immunocytochemistrywith a mouse monoclonal antibody against (x-smoothmuscle actin. C, Low magnification view of a keloidexplant (the explant was removed during the stain-ing process) ; scale bar= 1 mm. D, The region markedby a rectangle in Cwas observed with higher magni-fication ; scale bar= 100 lum.

110

SATO ET AL .

Figure 6 Collagen gel contractile capacity of keloid and normalskin fibroblasts . Fibroblasts were embedded in three-dimensionallattices of hydrated collagen gels, and the diameter of the gelswas measured during the culture period . Measurementwas donefor two sets of biopsies . For explanation of symbols, see Figure 2 .

5 to 10 days and remained almost unchanged in sizethereafter . In contrast, keloid explants decreased theirsize as early as 2 days of culture from the initial timeperiods:andcontinued to contract until around 20 dayswhen the size became approximately one third of theoriginal size (44% for keloid from patient No. 1 and22% for keloid from patient No. 2) . These results sug-gest that explants of keloid are more contractile thannormal skin explants .

Low magnification photomicrographs of explantstaken during culture clearly show that the growth offibroblast colonies formed by normal explants is muchmore rapid as compared with that of keloid explants(Figure 1) . The size of the fibroblast colonies was de-termined as a measure of the growth rate of the fibro-blasts (Figure 3) . Up to 15 days, no significant differ-ences in fibroblast colony size were observed between


thetwo types oftissue . Normal colonies started to growat a progressively higher rate than keloid coloniesthereafter . Normal explants on average produced fivetimes larger colonies at 25 days of culture comparedto keloid explants . Therefore, it appears that thegrowth potential of keloid fibroblasts is much lowerthan that of normal cells.

We happened to observe the presence ofcells whichwere outstandingly large in size as compared with themajority of cells in colonies formed by both normaland keloid explants (Figure 4) . We speculated thatthese enlarged cells might be myofibroblasts reportedby Gabbiani et al .7 which express a-smooth muscleactin as a component of their specific phenotype. Tocheck whether these cells were myofibroblasts or not,we performed immunostaining for a-smooth muscleactin. Enlarged cells were foundto be negative for thestaining (Figure 5) . Normal fibroblasts were faintlystained probably because of overexposure to the sec-ond antibody .

It was noticed by microscopic observations thatenlarged cells were found more frequently in keloid-explant cultures than in normal skin culture (Figure5) . Therefore, the enlarged cells were quantified forboth cultures by counting individually the cells whichwere negative to a-smooth muscle actin on photo-graphs as shown in Figure 5. Cultures of the two pa-tients yielded similar results . The appearance fre-quency of enlarged cells in keloid colonies was 4%,which was four times higher than that in normal cul-tures (1%) .

Collagen gel contraction by fibroblastsThe results shown in Figures 1 and 2 suggested thatthe contractile capacity of keloid fibroblasts exceedsthat ofnormal cells. Therefore, contractility ofthe cellswasquantitatively assayed in collagen lattices andwascompared between keloid and normal cells which hadbeen serially cultivated (Figure 6) . Unexpectedly, bothtypes of fibroblasts contracted collagen gels at a simi-lar rate .

Protein mapping of keloid cells by 2D-PAGETo know the phenotypic characteristics of keloid fi-broblasts at the protein level, we mapped proteins ongels of 2D-PAGE and compared them with those ofnormal cells . Fibroblasts at 20 to 25 passages wereused for protein mapping. As shown in Figure 7, nosignificant differences in the basic pattern of two-di-mensional protein maps were found between normaland keloid cells for both patients . A computer-assistedimage analyzer detected 151 spots of proteins on the

WOUND REPAIR AND REGENERATIONVOL. 4, NO. 1

Table 1. Identification of peptides on 2-D-PAGE gels

Spot No .

Peptide identified

2 Vimentin4 Vimentin5 Vimentin6 Vimentin7 Vimentin8 Actin13 Tropomyosin14 Tropomyosin16

Vimentin (fragment)17

Triosephosphate isomerase18

Vimentin (fragment)19

Vimentin (fragment)23 Stathmin26

Actin (fragment)28

Actin (fragment)29

Actin (fragment)

gel. Systematic identification by peptide fingerprint-ing with TOF-MS was applied to 31 protein spots onthe gels and 16 spots ofthem were successfully identi-fied as known proteins (Table 1, Figure 7) .

These maps were matched between paired nor-mal and keloid cells from the two patients with anaid of the image analyzer . Blue spots in Figure 8 represent matched proteins between normal and keloidcells, and therefore they are found commonly in thetwo types of cells. Red or green spots are unmatchedproteins : the reds are found in only keloid cells andthe greens in only normal cells. It is apparent thatblue color spots predominated on the maps, indicat-ing again that there is no significant qualitative al-teration in protein expression in keloid cells . How-ever, some quantitative changes in the level ofexpression were found. Fifty spots (33%) of proteinswere found to increase their amounts in keloid cellsas compared with normal cells and 34 spots (22.5%)of proteins were found to decrease . Of 16 spots iden-tified as known proteins, spot 23 (stathmin) showeda lower level of expression in keloid fibroblasts ascompared with normal cells.

DISCUSSIONKeloids result from excessive outgrowth of the dermis

at a site of injury which grows beyond the confines ofthe original wound site . Previous studies have at-

tempted to ascribe this morphologic abnormality ofkeloids to phenotypic alterations in keloid-forming fi-

SATO ET AL .

111

Figure 7 Two-dimensional gel electrophoresis of proteins of fibro-blasts . Preparative 2D-PAGE was performed for proteins obtainedfrom keloid (A) and normal (B) fibroblasts (patient No . 2) at the25th passage . Identical PAGE patterns were obtained from pa-tient No . 1 . Sixteen proteins among about 150 spots on gels wereidentified as known proteins with TOF-MS and were numbered .The names of these proteins are listed in Table 1 .

broblasts .l - lo However, there has not been a generallyaccepted answer to the question "Are there any phe-notypic alterations in keloid fibroblasts as comparedwith normal dermal fibroblasts?" The present studywasundertaken to biologically andbiochemically char-acterize keloid fibroblasts by comparing them withcorresponding characteristics of fibroblasts obtainedfrom normal skin near the keloid . The biopsy speci-mens we examined were diagnosed as keloid but notas hypertrophic scar. Keloids are different from hy-pertrophic scars in that keloids do not regress withtime, are difficult to revise surgically, and do not pro-voke scar contracturesis Therefore, chances to collectkeloid specimens for these analyses are rare .

We obtained such specimens together with nor-

mal skin samples from two individuals who had un-dergone plastic surgery procedures.

112

sATO ETA.

Figure 8 Computer-assisted matching of 2D-PAGE images between keloid and normal skin fibroblast proteins . Iden-tical matching results were obtained for patient Nos. 1 and 2. The photograph shown is from patient No . 2 . The 2D-PAGE gels were stained with Coomassie blue and scanned by the Bio Image Electrophoresis Analyzer and con-verted to video images . Red spots represent polypeptides expressed in only keloid fibroblasts; green spots representthose in only normal skin fibroblasts: blue spots represent matched proteins between normal and keloid cells .

In the present study, keloid fibroblasts were ex-amined for their morphology, growth potential, con-tractility, and protein expression . Two types of fibroblasts were prepared for the study: fibroblasts grownout from tissue explants (primary culture) and fibro-blasts under serial cultivations . It is generallythought that primary fibroblasts sustain character-istics expressed in vivo, whereas the cells tend to al-ter these phenotypic characteristics during serialcultivation. Keloid cells in the explant culture showedunique features in morphology, growth potential, andcontractility, which differ from normal fibroblasts .Both normal and keloid explants produced enlargedcells which are minor in the population of colony-forming cells. However, the rate of their appearancein colonies was much higher in keloid cultures (4%)than in normal cultures (1%) . Microscopic observa-tions of these peculiar cells suggest that they may bethe cells groupedas the Type 11 fibroblasts by Conwayet al .3 We examined the possibility that they aremyofibroblasts . However, the cells are immunocy-tochemically shown not to contain cytoplasmic (x-smooth muscle actin. The microscopic morphology ofthese cells is similar to the characteristics ofthe Type11 fibroblasts in that they are extremely flattened.3


The enlarged cells disappeared during serial cultiva-tion, suggesting that these cells have a high degreeof adhesiveness and/or a slower rate of division ascompared with the majority of normal fibroblasts .Further studies to correctly identify them have notbe done in the present study, and therefore their bio-logic significance in the generation of keloids is notknown. Nevertheless, they may play some importantroles in the pathologic changes of keloid tissues be-cause Conway et al .3 reported the presence of Type11 fibroblasts in the original keloid biopsies .

Using the explant culture of fibroblasts, we showthat the growth rate of keloid fibroblasts is lower com-pared with normal skin-derived fibroblasts . Harper'also reported the abnormal growth behavior of keloidfibroblasts in cultures . He used for his growth experi-ment serially propagated fibroblasts derived fromkeloid tissue and showed that keloid fibroblasts growat a rate which is approximately one half that of nor-mal age-, gender-, and race-matched control fibro-blasts . Conversely, a study reported by Diegelmannet al .' showed no differences in growth kinetics be-tween normal and keloid fibroblasts . These contradic-tions on the growth potential of keloid fibroblasts mayreflect differences in the assay system for growth ki-

WOUND REPAIR AND REGENERATIONVOL. 4, NO . 1

netics, regions from which keloid and normal skin bi-opsy samples were excised, and the degree of matura-tion in the keloid development after the injury. Atpresent, it is difficult to compare our results to thosecited here with regard to these parameters . However,it should be emphasized that we determined thegrowth kinetics of'keloid and normal fibroblasts fromthe explant culture, not of serially cultivated fibro-blasts . As emphasized repeatedly in the text, the ex-plant culture is thought to reproduce phenotypes ofcells in living tissues. Therefore, the present studystrongly suggests that keloid-forming fibroblasts showadecreased rate ofproliferation at least in some phaseof keloid development, although our study has a limi-tation that only two biopsy specimens diagnosed astrue keloid were available for the analysis . Furtherstudy is needed to show the nature of growth poten-tial of fibroblasts in relation to phase of keloid devel-opment .

Contractile capacity of fibroblasts is important tounderstand the mechanism of generation of tissuecontracture often found in hypertrophic scars . Weobserved that explants of keloid reduce their size rap-idly during culture and speculated that fibroblasts inthe keloid tissue have higher contractile ability. How-ever, it may be possible that cells in keloids showhigher collagenolytic activity which decompose col-lagen-rich keloid tissues. This hypothesis is unlikelybecause the explants did not lose their original andwell-organized form during the culture and show pro-gressively condensed appearance, which is differentfrom the changes we would expect from the degenera-tion of tissues. The contractile capacity as judged bydecrease in tissue size was not seen in the normal ex-plants .

In contrast to results obtained from explant cul-tures, no differences in the contractility were foundbetween keloid and normal cells which had been serially cultivated. It can be argued that keloid fibro-blasts lose during subculturing characteristics thatare related to thedecreased contractile capacity notedin the explant experiments. Recently, hypertrophicscar fibroblasts were shown to accelerate collagen gelcontraction and contract lattices at a significantlyfaster rate than do normal skin fibroblasts." Thisfinding led the authors to postulate that excessivescar contraction results from increased contractileforces generated by hypertrophic scar cells . There-fore, it seems that serially cultured hypertrophic scarfibroblasts retain their original features expressedin living tissues as far as the contractility is con-cerned . Currently, no studies have been reported on

SATO ET AL.

11 3

the performance of a systematic analysis of contrac-tile capacity of fibroblasts by comparing normal, hy-pertrophic, and keloid fibroblasts andusingthe samemethod of evaluation .

The present study made a thorough survey ofpro-teins present in keloid fibroblasts which are electro-phoretically separated on two-dimensional gels . About150 polypeptide spots were separated. To know differ-ences in quantity of these proteins between normaland keloid fibroblasts, we used a computer-assistedimage analyzer and succeeded in showing thatamounts of about one halfofthe detected proteins areincreased or decreased in cultured keloid fibroblastsas compared with those of normal fibroblasts .

As a trial to identify spots separated on gels, weapplied peptide fingerprinting analysis for 31 majorproteins using TOF-MS and succeeded in identifying16 spots as known proteins . Of these, stathmin wasthe only protein that showed different levels of ex-pression between normal and keloid fibroblasts.Stathmin is a ubiquitous cytosolic 19 kDa proteinwhich serves as a relay (for phosphorylation) for di-verse second messenger pathways . It has been shownthat stathmin is involved in the process of cell divi-sion and is expressed at a high level in the activelyproliferating cells . ' 8 Experiments with the explantculture clearly showed that growth potential of keloidfibroblasts is much lower compared with normal fi-broblasts. The decreased expression of stathmin inkeloid fibroblasts appears to coincide with their de-creased growth rate .

REFERENCES1. Wischer CW, Shetlar MR, ChvapilM. Hypertrophic scars and

keloids : a review and new concept concerning their origin . Scan-ning Microsc 1982 ;4 :1699-713.

2. Peacock EE, Madden JW Jr, Trier WC. Biologic basis for treat-ment of keloids and hypertrophic scars. South Med J1970 ;63 :755-60 .

3. Conway H, Gillette R, FindleyA. Observations on the behaviorofhuman keloids in vitro. Plast Reconstr Surg 1959 ;24:229-37 .

4. McCoy BJ, Galdun J, Cohen IK. Effects of density and cellularaging on collagen synthesis and growth kinetics in keloid andnormal skin fibroblasts . In Vitro 1982 ;18:79-86.

5. Diegelmann RF, Cohen IK, McCoy BJ . Growth kinetics and col-lagen synthesis of normal skin, normal scar and keloid fibro-blasts in vitro. J Cell Physiol 1979 ;98:341-6 .

6. Cohen IK, Keiser HR, SjoerdsmaA. Collagen synthesis in hu-man keloid and hypertrophic scar . Surg Forum 1971 ;22:488-9.

7. Gabbiani G, Ryan GB, MajnoG. Presence ofmodified fibroblastsin granulation tissue and possible role in wound contracture .Experientia 1971 ;27:549 .

S. Harper RA. Keloid fibroblasts in culture : abnormal growthbehaviour and altered response to the epidermal growth factor .Cell Biol Int Rep 1989;13:325-35 .

9. McGrouther DA . Hypertrophic or keloid scars?Eye 1994 ;8 :200-3 .

114

SATO ET AL .

10. Craig RDP, Schofield JD, Jackson SS . Collagen biosynthesis innormal human skin, normal and hypertrophic scar and keloid.Eur J Clin Invest 1975 ;5 :69-79.

11 . Asaga H, Kikuchi S, Yoshizato K. Collagen gel contraction byfibroblasts requires cellular fibronectin but not plasmafibronectin. Exp Cell Res 1991;193:167-74 .

12. Koseki N, Yoshizato K. Collagen-induced changes in the pat-tern of protein synthesis of fibroblasts . Cell Adhes Commun1994 ;1 :355-66 .

13. O'Farrell PH . High resolution two-dimensional electrophoresisof proteins . J Biol Chem 1975;250 :4007-21 .

14. Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C,Watanabe C. Identifyingproteins from two-dimensional gels bymolecular mass searching of peptide fragments in protein se-quence database. Proc Natl Arad SciUSA1993;90:5011-5 .

O N THE MOVE?1-

end us your new address at least six weeks ead

Don't miss a single issue of the journal! To ensure prompt service when you change your address,please photocopy and complete the form below.

Please send your change ofaddress notification at least six weeks before your move to ensure continued service.We regret we cannot guarantee replacement of issues missed due to late notification.

JOURNAL TITLE:Fill in the title of thejournal here .

OLD ADDRESS:

15. Sutton CW, Pemberton KS, Cottrell JS, Corbett JM, WheelerCH, Dunn MJ, PappinDJ. Identification ofmyocardial proteinsfrom two-dimensionalgels bypeptide mass fingerprinting. Elec-trophoresis 1995;16:308-16 .

16. Ehrlich HP, Desmouliere A, Diegelmann RF, Cohen IK,Compton CC, Garner WL, Kapanci Y, Gabbiani G. Morphologi-cal and immunochemical differences between keloid and hyper-trophic scar. Am J Pathol 1994 ;145 :105-13 .

17. Garner WL, Rittenberg T, Ehrlich HP, Karmiol S, RodriguezJL, Smith DJ Jr, Phan SH . Hypertrophic scar fibroblasts accel-erate collagen gel contraction. WOUND REP REG 1995 ;3 :185-91 .

18. Rowlands DC, Williams A, Jones NA, Guest SS, Reynolds GM,Barber PC, BrownG. Stathmin expression is a feature of prolif-erating cells of most, if not all, cell linages . Lab Invest1995 ;72:100-13.

NEW ADDRESS:


Affix the address label from a recent issue of the journalhere. Clearly print your new

Name

Address

City/State/ZIP

address here .

COPY AND MAIL THIS FORM TO: OR FAX TO: OR PHONE:Journal Subscription Services 314-432-1158 1-800-453-4351Mosby-Year Book, Inc. Outside the U.S ., call11830 Westline Industrial Dr. KFA Mosby 314-453-4351St. Louis, MO 63146-3318

characteristics of growth, morphology, contractility, and protein expression of fibroblasts derived...

Documents