- Email: [email protected]
Keloid fibroblasts in culture: Abnormal growth behaviour and altered response to the epidermal growth factor
Cell Biology
international
KELOID BEHAVIOUR
Reports,
FIBROBLASTS AND ALTERED
Vol. 13, No. 4, April
IN-CULTURE: RESPONSE
1989
ABNORMAL
325
GROWTH
TO THE EPIDERMAL
GROWTH
FACTOR
Robert
A.
Harper
Connective Tissue Research Institute, University Science Center, 3624 Market Street, Philadelphia. Pennsylvania 19104.
City
ABSTRACT
fibroblasts were propagated in culture and their Keloid proliferative behaviour and response to the Epidermal Factor were studied. Growth Keloid fibroblasts (EGF) at a rate which was approximately one-half that of grew and race matched control fibroblasts. normal sex age, were Keloid fibroblasts stimulated to grow in the presence of but to a lesser degree than EGF (long/ml), normal the control fibroblasts. Scatchard analysis of binding data obtained 1*51-labeled the using EGF in binding affinity or receptor revealed no difference numbers between keloid and normal fibroblasts. INTRODUCTION
A keloid is a localised area of excessive fibrous tissue arising at proliferation usually the site of injury, form of trauma ( Murray et surgical wound, burn or other sites of involvement are the ear 1981). Frequent al., lobes, back and abdomen. Keloids are most wpe 1: frequently encountered in the darker pigmented races, but have been reported in virtually every race. Histologically mature keloids are rich in extracellular matrix, including colloids and proteoglycans (Lever and 1983). During early formation of the Schaumberg-Lever, fibroblasts are numerous, but in older lesions keloid, they are relatively few. utilising " in Conflicting reports exist fibroblasts mechanism of extracellular vitroV to elucidate the matrix production in keloids. Abergel et al. (1985) which it was shown that, in a published a report in keloid cell cultures studied, there was an majority of increase in procollagen production. In addition, in some 0309-1651/89/04032Er1
l/$03.00/0
@ 1969 Academic
Press Ltd.
326
Cell Biology
International
Reports,
Vol. 13, No. 4, April
1989
cultures were able to show that of the the authors degradation of newly synthesised collagen polypeptides was below the range of normal controls. More recently, however, Ala-Kokka et a1.(1987) reported the synthesis of normal amounts of collagen from keloid fibroblasts. Previous studies have indicated that keloid fibroblasts in culture demonstrate normal growth behaviour (Diegelmann et al., but are affected by 1979), extracellular factors in a different manner than normal fibroblasts 1972, 1987). Since (Russell et al., Epidermal Growth Factor (EGF) is known to stimulate cell growth (Carpenter and Cohen., and to 1979) affect collagen (Kumegawa et al., 1981) and proteoglycan biosynthesis (Lenbach, 1976), we have chosen to study the behaviour of keloid fibroblasts in EGF. MATERIALS AND METHODS Clinical Material: Tissue specimens were taken from 2 patients with keloids. The keloid tissue was surgically removed under local anaesthesia after obtaining informed consent. The patients were black females aged 22 and 29 years. Fibroblasts from patient #l ( 22 yr. old) were used for the data shown in Table 1 and Figures 1 and 3.Fibroblasts from patient #2 ( 29 yr. old) were used for the data appearing in Figure 2. The lesions were located on the earlobe and were diagnosed as keloids on the basis of their clinical appearance and histopathology. "Normal" adjacent tissue to the keloid from patient #2 obtained in was order to establish from fibroblasts uninvolved skin of keloids. Control skin was obtained from a normal sex, age and racematched patient who had undergone cosmetic surgery. These were tissues used to establish fibroblast cultures. Cell Culture: Primary cultures of skin fibroblasts were established as previously described (Harper and Burgoon, 1982). All fibroblast strains were maintained in Eagle's minimal essential medium containing 6% fetal calf serum, 100 U/ml penicillin, 100 pg/ml streptomycin and 30 U/ml nystatin. Culture medium was changed every third day. were Cells propagated in a high humidity incubator at 37OC in an atmosphere of 5% CO, and air. After 21 days in primary culture, fibroblast the outgrowth was trypsinized and subcultured. Subsequent trypsinations were performed every two weeks. For all experiments, the cells were used at less than 6 passages with trypsin and
Cell Biology
had
International
accumulated
Reports,
no more
Vol. 13, No. 4, April
than
12 population
1989
327
doublings.
Epidermal Factor: Growth Purified mouse EGF and 1251-labeled EGF were generously provided by Dr. Richard Department of Savage', Jr., Biochemistry, Temple University School of Medicine, Philadelphia,, PA. The EGF had been isolated from the submaxillary glands of adult of Savage and Cohen (1972). male mice by the method Purified radioiodinated EGF was with carrier-free Na1251 using the procedure of Moriarity and Savage (1980): Growth Studies: Fibroblasts were trypsinized and 5~10~ seeded into 100mm tissue culture dishes (Co-Star) cells 5.0 ml of culture medium. Twenty-four hours containing after seeding, EGF was added to a final concentration of long/ml. On the 4th, 7th and 11th day after seeding, the used culture medium was removed and fresh culture medium Control dishes received no EGF. At plus EGF was added. two dishes were removed from each various time points, were trypsinized the cells and counted in a group, Each point on the curves represents the Coulter counter. cell count between duplicate dishes with a average of less than 10%. Cell viability, as estimated variance by exclusion of trypan blue dye, was greater than 95% at all time points. 1251-EGF binding studies were conducted Binding Studies: confluent monolayers fibroblasts in using of 24-multiwell culture tissue plates (Co-Star) as 1981). The previously described (Savage and Harper, to 3 specific radioactivity of the 1251-EGF was adjusted by the addition of non-labeled EGF and the final uCi/w concentration of EGF incubated with the cells ranged 0.08 to 1.5nM. Non-specific binding was determined from in the presence of lOOO-fold excess of unlabeled EGF and than 10% of cell bound was always less the total radioactivity. The incubation carried out for 90 was data were analysed by the method of min. at 4OC. The Scatchard (1949) in order to determine the concentration for half binding and the of EGF required maximal apparent number of receptors. RESULTS The growth response of keloid and normal fibroblasts "in depicted in Figure 1. Keloid vitro" to EGF is fibroblasts in the absence of EGF exhibited a slower normal rate of than skin fibroblasts, and at growth confluency, achieved a final cell density 50% less than
Cell Biology
32%
International
Reports,
Vol. 73, No. 4, April
1989
normal fibroblasts. In the presence of EGF (long/ml), rate of keloid fibroblast growth was increased with the cells achieving a 2-fold increase in cell number at the saturation However, density. the sex and race age, control fibroblast matched culture contained 3-4 fold more cells at saturation density in the presence of EGF. I
I
I
I
10 9
KELOID + EGF
KELOID
3
Figure vitro"
of 1. Growth in the presence
6
10 Time (days)
keloid and normal of EGF ( long/ml.).
14
fibroblasts
"in
and the surrounding "uninvolved" Tissue from a keloid and fibroblast skin was taken from a second patient cultures established. The growth response of these cells EGF is Figure 2. As seen with to depicted in the from patient #l (Figure l), the keloid fibroblasts fibroblasts grew very poorly, with only a doubling of in 13 days. The "non-involved" fibroblasts cell number divided at a faster rate than the keloid fibroblasts resulting in a 3-fold increase in cell number by day 13.
Cell Biology
International
Reports,
Vol. 73, No. 4, April
In the fibroblasts saturation untreated
presence of EGF, both keloid and were stimulated to proliferate density of approximately cultures.
“r----l 3l-
NON-INVOLVED
1989
329
"non-involved" and reached a 2-fold over
+ EGF
-I
KELOlD + EGF
NON-INVOLVED
3
Figure 2. Ilin vitro"
6 Days in Culture
Growth of keloid and "uninvolved in the presence of EGF (long/ml).
fibroblasts
In order to determine whether the lack of response to keloid fibroblasts EGF by was due to a change in EGF binding affinity or receptor number, binding experiments were carried out using 1251-labeled EGF. Figure 3 shows a typical binding profile for normal and keloid fibroblasts. A Scatchard type analysis of the binding data ( see inset) revealed half saturation occurring at 1.49nM for the normal fibroblasts and 2.4nM for the keloid fibroblasts with 14,200 and 12,800 apparent binding sites per cell, respectively. These values fall within the range of values we have previously published
330
Cell Biology
International
Reports,
Vol. 73, No. 4, April
1989
for normal cells (Schaudies et al., 1985). Scatchard of other binding experiments conducted at 37OC analysis revealed no differences in binding kinetics between normal and keloid fibroblasts (data not shown). 125 I - Labeled
EGF (r&l) 1.5
0.75 I
6
t
6
NORMAL
KELOD
FIBROBLASTS
FMOBLASTS
t 5 At
’
12345676 ’ 25 I - Labeled
Figure
fibroblasts. presented
3. Binding of lZ51-EGF Scatchard type in the insets.
9
10
EGF hghl)
to keloid analysis
and normal skin of the data is
DISCUSSION
It has been assumed from previous studies that keloid exhibit fibroblasts normal growth behaviour in culture (Diegelmann et al., 19791, but have an altered response to various extracellular factors such as hydrocortisone and histamine (Russell et al., 1977, 1982). Our studies reveal for first time, a difference in the growth the behaviour of keloid fibroblasts in culture. The keloid fibroblast strains we established "in vitro" from two
Cell Biology
International
Reports,
Vol. 13, No. 4, April
1989
331
patients obtained a final cell density lower than the life normal control fibroblasts. When the "in vitro" was compared between keloid fibroblasts (patient span normal fibroblasts, the cumulative population and #l) doublings acquired by keloid cell strain at the senescence was 31% less than those of the normal control fibroblasts (data not shown). Differences in growth behaviour also existed between the keloid fibroblasts propagated from the and the surrounding "non-involved" area. At the end of six days, the cell density of keloid and "non-involved" fibroblast cultures was the same (Figure 2). However, between six thirteen days a divergence in growth appeared, and in cell number of the resulting in a 50% increase "non-involved" fibroblasts over the keloid cultures. The reasons for this difference in growth rates between normal and keloid fibroblasts are unknown at this time. However, this cannot be accounted for by a difference in efficiency. A slightly greater plating plating efficiency was found for the keloid fibroblasts (data not shown). It is possible that the higher plating efficiency of the keloid fibroblasts is due to greater synthesis of extracellular matrix proteins. The reasons differ from previous studies concerning the data our growth behaviour of keloid fibroblasts is not known. Our "in vitro" under similar were cell strains propagated conditions as reported for the earlier studies and both studies utilised low passage cells. One variation between studies could be the maturity of the keloid. It might be that fibroblasts from a very young keloid could different growth properties in culture than exhibit fibroblasts from an older, more fibrotic keloid. It is formation of keloid known that during the early are numerous, but in older lesions they are fibroblasts Fibroblasts from a mature 1980). relatively few (Smith, have undergone more population doublings keloid could " in viva" which could account for poor growth the "in vitro" life span observed in behaviour and reduced our studies. possibility is that a different population of Another which have exist in keloids "fibroblast-like" cells normal characteristics than different growth idea seems reasonable since a recent fibroblasts. This revealed some keloids are electron microscopic study "myofibroblasts" (James et al., composed entirely of 1980). Myofibroblasts are morphologically distinct cells fibroblasts and smooth with characteristics of both muscle cells. Whether myofibroblasts were present in our
Cell Biology
cultures
is
unknown
International
at
this
Reports,
Vol. 73, No. 4, April
1989
time.
depicted in Figures 1 and 2 show clearly an The data fibroblasts to EGF. At abnormal response of keloid keloid fibroblasts reach a maximum cell confluence, much lower than normal fibroblasts or density that is fibroblasts obtained from the "non-involved" area What is possibly more significant surrounding a keloid. however, is the comparison of the rate of cell growth and cell density achieved at day 6. At this time period, keloid fibroblasts in the presence of EGF have only normal grown to a cell density equal to that of fibroblasts in the absence of EGF (Figure 1). Our findings are in agreement with Russel et a1,(1988), who have reported that normal fibroblasts incorporate more 3H-thymidine in the presence of EGF than do fibroblasts from keloids. Using fibroblasts from a second keloid (Figure 2). essentially the same results are seen when keloid to "non-involved" fibroblasts. At day comparing keloid have not responded to EGF while fibroblasts 6, fibroblasts from the "non-involved" area of the the keloid patient have responded to EGF (2.5-fold increase in number over cell untreated cells). Whether the fibroblasts from the "non-involved" area surrounding the keloid abnormal growth characteristics "in demonstrate vitro' or respond differently to EGF is uncertain at this time. While for the abnormal response of keloid the reasons fibroblasts to EGF are unknown, one could speculate that the keloid fibroblasts are secreting excessive amounts of collagen or some other extracellular matrix protein which could alter the response of the fibroblast to EGF. In order to answer this question, experiments are needed the synthesis of extracellular matrix to correlate proteins with the growth behaviour of keloid fibroblasts their response and to EGF utilising a large number of fibroblast strains established from keloids of known maturity. As mentioned above, the fibroblasts from the keloid have could undergone more population doublings "in vivoI' than the normal controls. If this is the case, they could be less responsive to EGF. Our laboratory has observed routinely that normal adult human skin fibroblasts become less responsive to the growth stimulation of EGF as the population doublings of the cells increase "in vitro", even though the cells exhibit normal EGF binding kinetics (unpublished results). Another possibility EGF-receptor binding such as EGF stimulated
is that a defect event exists in keloid phosphorylation,"down
in a post fibroblasts, regulation"
Cell Biology
of of
International
Reports,
Vol. 13, No. 4, April
the EGF-receptor complex, EGF. These possibilities
or are
intracellular currently
1989
333
being
processing pursued.
ACKNOWLEDGMENTS The author extremely the expert
would like to thank Dr Robert Alper for helpful suggestions and Maryann Mason typing of the submitted manuscript.
his for
REFERENCES
Abergel, R-P., Pizzurro, D., Meeker, C.A., Lask, G., Matsuoka, L-Y., Minor,R.R., Chu, H-L. and Uitto, J. Biochemical (1985) composition of the connective tissue in keloids metabolism in keloid Derm. 84: 384-390
Ala-Kokko,
L.,
Collagen collagen collagen fibroblast 238-244.
Rintala,
G.
Factor.
Ann.
Diegelmann,
and Rev.
R-F.,
Growth kinetics skin, normal Physiol. J. Cell
R.A.
of retinoic like cells skin. Cell
James,
W.D.,
Cohen,
S.
(1979)
Biochem.
48:
193-216.
Cohen, and scar 98:
E.R.
collagen Invest.
(1987)
1-K.
Epidermal
and McCoy, synthesis fibroblasts
collagen and keloid 341-346.
B.J. of in
Growth
(1979) normal vitro.
effects T. (1982) Differential acid on the growth of normal fibroblast" in vitro" from human, swine and rabbit Biol. Inter. Reps. 6: 163-170.
and Burgoon,
Bescanceney,
ultrastructure 3:
A. and Savolainen,
J.
expression in keloids: Analysis of gene metabolism and Type 1,111, IV and V prokeloid mRNAs in keloid tissue and J. Invest. Dermatol. 89: cultures.
Carpenter,
Harper,
and analysis of fibroblast cultures.
of
C.D.
a keloid.
and Odum, R.B. J.
Amer.
Acad.
(1980) The Dermatol.
50-57.
Kumegawa, M., Hatakeyama,
Yajima, T., Hiramatsu, K. and Namba, M. (1981)
stimulates Factor derived epithelial Acta 675: 305-308.
collagen clone cells.
M.,
Ikeda,
E.,
Epidermal Growth liversynthesis in Biochem. Biophys.
Cell Biology
Lembach, extracellular stimulation Epidermal
International
K.J.
(1976) accumulation of quiescent, Growth Factor.
Reports,
Vol. 73, No. 4, April
7989
synthesis Enhanced and of hyaluronic acid during human fibroblasts by mouse J. Cell. Physiol. 89:
277-288.
Lever, W.F. and SchaumburgLever, G. tissue. In: Histopathology fibrous Lippincott, Philadelphia. 577-578, Moriarity, Epidermal cells in 203:
(1983) Tumours of of the skin. pp.
Interaction of D.M. and Savage, C.R. (1980) Growth Factor with adult rat parenchymal primary culture. Arch. Biochem. Biophys.
505-518.
Murray, Keloids:
J-C., a
Pollack, review.
and Pinnell, S.V. J. Amer. Acad.
S.R. (1981) Dermatol. 4:
461-470.
Russell, J-D., Russell, effect of histamine fibroblasts isolated Physiol. 93: J. Cell.
S.B.
and Trupin, K.M. (1977) The on the growth of cultured from normal and keloid tissue. 389-394.
Russell, J.D. and Trupin, Russell, S-B., Alteration of amino acid transport cortisone: different effects in human derived from normal skin and keloid. J. 257: 9525- 9531. Russell, S-R., Trupin, K-M., Russell, J.D. and Trupin, growth-factor requirement fibroblasts for may account 85: 587-591. Nat. Acad. Sci. Savage, C.R. Factor and 7609-7611.
and Cohen, S. a new derivative.
J.S. (1982) by hydrofibroblasts Biol. Chem.
Rodriguez-Eaton, S., J.S. (1988) Reduced keloid-derived of growth. tumour Proc. (1972) J.
Epidermal Growth Biol. Chem. 247:
R.A. (1981) Human Epidermal Savage, C.R. and Harper, Growth Factor/ Urogastrone: Rapid purification procedure partial characterisation. and Analyt. Biochem. 111: 195-202. Scatchard, G. (1949) The attractions of proteins small molecules and ions. Ann. New York Acad. 51: 660-675.
for Sci.
Cell Biology
International
Reports,
Vol. 13, No. 4, April
335
1989
Schaudies, R-P., Harper, R.A. and Savage, C.R. (1985) 1251-EGF binding to responsive and non-responsive cells in culture: LOSS of cell associated radioactivity relates to growth induction. J. Cell Physiol. 124: 493-498. (1980) Tumours of Smith, J.L. (eds.). Helwig and F.K. Mostofi Williams and Wilkins, Baltimore.
Paper
received
12.5.88
the Corium. In: E.B. The skin. pp 553-557,
Paper
accepted
12.12.88
international
KELOID BEHAVIOUR
Reports,
FIBROBLASTS AND ALTERED
Vol. 13, No. 4, April
IN-CULTURE: RESPONSE
1989
ABNORMAL
325
GROWTH
TO THE EPIDERMAL
GROWTH
FACTOR
Robert
A.
Harper
Connective Tissue Research Institute, University Science Center, 3624 Market Street, Philadelphia. Pennsylvania 19104.
City
ABSTRACT
fibroblasts were propagated in culture and their Keloid proliferative behaviour and response to the Epidermal Factor were studied. Growth Keloid fibroblasts (EGF) at a rate which was approximately one-half that of grew and race matched control fibroblasts. normal sex age, were Keloid fibroblasts stimulated to grow in the presence of but to a lesser degree than EGF (long/ml), normal the control fibroblasts. Scatchard analysis of binding data obtained 1*51-labeled the using EGF in binding affinity or receptor revealed no difference numbers between keloid and normal fibroblasts. INTRODUCTION
A keloid is a localised area of excessive fibrous tissue arising at proliferation usually the site of injury, form of trauma ( Murray et surgical wound, burn or other sites of involvement are the ear 1981). Frequent al., lobes, back and abdomen. Keloids are most wpe 1: frequently encountered in the darker pigmented races, but have been reported in virtually every race. Histologically mature keloids are rich in extracellular matrix, including colloids and proteoglycans (Lever and 1983). During early formation of the Schaumberg-Lever, fibroblasts are numerous, but in older lesions keloid, they are relatively few. utilising " in Conflicting reports exist fibroblasts mechanism of extracellular vitroV to elucidate the matrix production in keloids. Abergel et al. (1985) which it was shown that, in a published a report in keloid cell cultures studied, there was an majority of increase in procollagen production. In addition, in some 0309-1651/89/04032Er1
l/$03.00/0
@ 1969 Academic
Press Ltd.
326
Cell Biology
International
Reports,
Vol. 13, No. 4, April
1989
cultures were able to show that of the the authors degradation of newly synthesised collagen polypeptides was below the range of normal controls. More recently, however, Ala-Kokka et a1.(1987) reported the synthesis of normal amounts of collagen from keloid fibroblasts. Previous studies have indicated that keloid fibroblasts in culture demonstrate normal growth behaviour (Diegelmann et al., but are affected by 1979), extracellular factors in a different manner than normal fibroblasts 1972, 1987). Since (Russell et al., Epidermal Growth Factor (EGF) is known to stimulate cell growth (Carpenter and Cohen., and to 1979) affect collagen (Kumegawa et al., 1981) and proteoglycan biosynthesis (Lenbach, 1976), we have chosen to study the behaviour of keloid fibroblasts in EGF. MATERIALS AND METHODS Clinical Material: Tissue specimens were taken from 2 patients with keloids. The keloid tissue was surgically removed under local anaesthesia after obtaining informed consent. The patients were black females aged 22 and 29 years. Fibroblasts from patient #l ( 22 yr. old) were used for the data shown in Table 1 and Figures 1 and 3.Fibroblasts from patient #2 ( 29 yr. old) were used for the data appearing in Figure 2. The lesions were located on the earlobe and were diagnosed as keloids on the basis of their clinical appearance and histopathology. "Normal" adjacent tissue to the keloid from patient #2 obtained in was order to establish from fibroblasts uninvolved skin of keloids. Control skin was obtained from a normal sex, age and racematched patient who had undergone cosmetic surgery. These were tissues used to establish fibroblast cultures. Cell Culture: Primary cultures of skin fibroblasts were established as previously described (Harper and Burgoon, 1982). All fibroblast strains were maintained in Eagle's minimal essential medium containing 6% fetal calf serum, 100 U/ml penicillin, 100 pg/ml streptomycin and 30 U/ml nystatin. Culture medium was changed every third day. were Cells propagated in a high humidity incubator at 37OC in an atmosphere of 5% CO, and air. After 21 days in primary culture, fibroblast the outgrowth was trypsinized and subcultured. Subsequent trypsinations were performed every two weeks. For all experiments, the cells were used at less than 6 passages with trypsin and
Cell Biology
had
International
accumulated
Reports,
no more
Vol. 13, No. 4, April
than
12 population
1989
327
doublings.
Epidermal Factor: Growth Purified mouse EGF and 1251-labeled EGF were generously provided by Dr. Richard Department of Savage', Jr., Biochemistry, Temple University School of Medicine, Philadelphia,, PA. The EGF had been isolated from the submaxillary glands of adult of Savage and Cohen (1972). male mice by the method Purified radioiodinated EGF was with carrier-free Na1251 using the procedure of Moriarity and Savage (1980): Growth Studies: Fibroblasts were trypsinized and 5~10~ seeded into 100mm tissue culture dishes (Co-Star) cells 5.0 ml of culture medium. Twenty-four hours containing after seeding, EGF was added to a final concentration of long/ml. On the 4th, 7th and 11th day after seeding, the used culture medium was removed and fresh culture medium Control dishes received no EGF. At plus EGF was added. two dishes were removed from each various time points, were trypsinized the cells and counted in a group, Each point on the curves represents the Coulter counter. cell count between duplicate dishes with a average of less than 10%. Cell viability, as estimated variance by exclusion of trypan blue dye, was greater than 95% at all time points. 1251-EGF binding studies were conducted Binding Studies: confluent monolayers fibroblasts in using of 24-multiwell culture tissue plates (Co-Star) as 1981). The previously described (Savage and Harper, to 3 specific radioactivity of the 1251-EGF was adjusted by the addition of non-labeled EGF and the final uCi/w concentration of EGF incubated with the cells ranged 0.08 to 1.5nM. Non-specific binding was determined from in the presence of lOOO-fold excess of unlabeled EGF and than 10% of cell bound was always less the total radioactivity. The incubation carried out for 90 was data were analysed by the method of min. at 4OC. The Scatchard (1949) in order to determine the concentration for half binding and the of EGF required maximal apparent number of receptors. RESULTS The growth response of keloid and normal fibroblasts "in depicted in Figure 1. Keloid vitro" to EGF is fibroblasts in the absence of EGF exhibited a slower normal rate of than skin fibroblasts, and at growth confluency, achieved a final cell density 50% less than
Cell Biology
32%
International
Reports,
Vol. 73, No. 4, April
1989
normal fibroblasts. In the presence of EGF (long/ml), rate of keloid fibroblast growth was increased with the cells achieving a 2-fold increase in cell number at the saturation However, density. the sex and race age, control fibroblast matched culture contained 3-4 fold more cells at saturation density in the presence of EGF. I
I
I
I
10 9
KELOID + EGF
KELOID
3
Figure vitro"
of 1. Growth in the presence
6
10 Time (days)
keloid and normal of EGF ( long/ml.).
14
fibroblasts
"in
and the surrounding "uninvolved" Tissue from a keloid and fibroblast skin was taken from a second patient cultures established. The growth response of these cells EGF is Figure 2. As seen with to depicted in the from patient #l (Figure l), the keloid fibroblasts fibroblasts grew very poorly, with only a doubling of in 13 days. The "non-involved" fibroblasts cell number divided at a faster rate than the keloid fibroblasts resulting in a 3-fold increase in cell number by day 13.
Cell Biology
International
Reports,
Vol. 73, No. 4, April
In the fibroblasts saturation untreated
presence of EGF, both keloid and were stimulated to proliferate density of approximately cultures.
“r----l 3l-
NON-INVOLVED
1989
329
"non-involved" and reached a 2-fold over
+ EGF
-I
KELOlD + EGF
NON-INVOLVED
3
Figure 2. Ilin vitro"
6 Days in Culture
Growth of keloid and "uninvolved in the presence of EGF (long/ml).
fibroblasts
In order to determine whether the lack of response to keloid fibroblasts EGF by was due to a change in EGF binding affinity or receptor number, binding experiments were carried out using 1251-labeled EGF. Figure 3 shows a typical binding profile for normal and keloid fibroblasts. A Scatchard type analysis of the binding data ( see inset) revealed half saturation occurring at 1.49nM for the normal fibroblasts and 2.4nM for the keloid fibroblasts with 14,200 and 12,800 apparent binding sites per cell, respectively. These values fall within the range of values we have previously published
330
Cell Biology
International
Reports,
Vol. 73, No. 4, April
1989
for normal cells (Schaudies et al., 1985). Scatchard of other binding experiments conducted at 37OC analysis revealed no differences in binding kinetics between normal and keloid fibroblasts (data not shown). 125 I - Labeled
EGF (r&l) 1.5
0.75 I
6
t
6
NORMAL
KELOD
FIBROBLASTS
FMOBLASTS
t 5 At
’
12345676 ’ 25 I - Labeled
Figure
fibroblasts. presented
3. Binding of lZ51-EGF Scatchard type in the insets.
9
10
EGF hghl)
to keloid analysis
and normal skin of the data is
DISCUSSION
It has been assumed from previous studies that keloid exhibit fibroblasts normal growth behaviour in culture (Diegelmann et al., 19791, but have an altered response to various extracellular factors such as hydrocortisone and histamine (Russell et al., 1977, 1982). Our studies reveal for first time, a difference in the growth the behaviour of keloid fibroblasts in culture. The keloid fibroblast strains we established "in vitro" from two
Cell Biology
International
Reports,
Vol. 13, No. 4, April
1989
331
patients obtained a final cell density lower than the life normal control fibroblasts. When the "in vitro" was compared between keloid fibroblasts (patient span normal fibroblasts, the cumulative population and #l) doublings acquired by keloid cell strain at the senescence was 31% less than those of the normal control fibroblasts (data not shown). Differences in growth behaviour also existed between the keloid fibroblasts propagated from the and the surrounding "non-involved" area. At the end of six days, the cell density of keloid and "non-involved" fibroblast cultures was the same (Figure 2). However, between six thirteen days a divergence in growth appeared, and in cell number of the resulting in a 50% increase "non-involved" fibroblasts over the keloid cultures. The reasons for this difference in growth rates between normal and keloid fibroblasts are unknown at this time. However, this cannot be accounted for by a difference in efficiency. A slightly greater plating plating efficiency was found for the keloid fibroblasts (data not shown). It is possible that the higher plating efficiency of the keloid fibroblasts is due to greater synthesis of extracellular matrix proteins. The reasons differ from previous studies concerning the data our growth behaviour of keloid fibroblasts is not known. Our "in vitro" under similar were cell strains propagated conditions as reported for the earlier studies and both studies utilised low passage cells. One variation between studies could be the maturity of the keloid. It might be that fibroblasts from a very young keloid could different growth properties in culture than exhibit fibroblasts from an older, more fibrotic keloid. It is formation of keloid known that during the early are numerous, but in older lesions they are fibroblasts Fibroblasts from a mature 1980). relatively few (Smith, have undergone more population doublings keloid could " in viva" which could account for poor growth the "in vitro" life span observed in behaviour and reduced our studies. possibility is that a different population of Another which have exist in keloids "fibroblast-like" cells normal characteristics than different growth idea seems reasonable since a recent fibroblasts. This revealed some keloids are electron microscopic study "myofibroblasts" (James et al., composed entirely of 1980). Myofibroblasts are morphologically distinct cells fibroblasts and smooth with characteristics of both muscle cells. Whether myofibroblasts were present in our
Cell Biology
cultures
is
unknown
International
at
this
Reports,
Vol. 73, No. 4, April
1989
time.
depicted in Figures 1 and 2 show clearly an The data fibroblasts to EGF. At abnormal response of keloid keloid fibroblasts reach a maximum cell confluence, much lower than normal fibroblasts or density that is fibroblasts obtained from the "non-involved" area What is possibly more significant surrounding a keloid. however, is the comparison of the rate of cell growth and cell density achieved at day 6. At this time period, keloid fibroblasts in the presence of EGF have only normal grown to a cell density equal to that of fibroblasts in the absence of EGF (Figure 1). Our findings are in agreement with Russel et a1,(1988), who have reported that normal fibroblasts incorporate more 3H-thymidine in the presence of EGF than do fibroblasts from keloids. Using fibroblasts from a second keloid (Figure 2). essentially the same results are seen when keloid to "non-involved" fibroblasts. At day comparing keloid have not responded to EGF while fibroblasts 6, fibroblasts from the "non-involved" area of the the keloid patient have responded to EGF (2.5-fold increase in number over cell untreated cells). Whether the fibroblasts from the "non-involved" area surrounding the keloid abnormal growth characteristics "in demonstrate vitro' or respond differently to EGF is uncertain at this time. While for the abnormal response of keloid the reasons fibroblasts to EGF are unknown, one could speculate that the keloid fibroblasts are secreting excessive amounts of collagen or some other extracellular matrix protein which could alter the response of the fibroblast to EGF. In order to answer this question, experiments are needed the synthesis of extracellular matrix to correlate proteins with the growth behaviour of keloid fibroblasts their response and to EGF utilising a large number of fibroblast strains established from keloids of known maturity. As mentioned above, the fibroblasts from the keloid have could undergone more population doublings "in vivoI' than the normal controls. If this is the case, they could be less responsive to EGF. Our laboratory has observed routinely that normal adult human skin fibroblasts become less responsive to the growth stimulation of EGF as the population doublings of the cells increase "in vitro", even though the cells exhibit normal EGF binding kinetics (unpublished results). Another possibility EGF-receptor binding such as EGF stimulated
is that a defect event exists in keloid phosphorylation,"down
in a post fibroblasts, regulation"
Cell Biology
of of
International
Reports,
Vol. 13, No. 4, April
the EGF-receptor complex, EGF. These possibilities
or are
intracellular currently
1989
333
being
processing pursued.
ACKNOWLEDGMENTS The author extremely the expert
would like to thank Dr Robert Alper for helpful suggestions and Maryann Mason typing of the submitted manuscript.
his for
REFERENCES
Abergel, R-P., Pizzurro, D., Meeker, C.A., Lask, G., Matsuoka, L-Y., Minor,R.R., Chu, H-L. and Uitto, J. Biochemical (1985) composition of the connective tissue in keloids metabolism in keloid Derm. 84: 384-390
Ala-Kokko,
L.,
Collagen collagen collagen fibroblast 238-244.
Rintala,
G.
Factor.
Ann.
Diegelmann,
and Rev.
R-F.,
Growth kinetics skin, normal Physiol. J. Cell
R.A.
of retinoic like cells skin. Cell
James,
W.D.,
Cohen,
S.
(1979)
Biochem.
48:
193-216.
Cohen, and scar 98:
E.R.
collagen Invest.
(1987)
1-K.
Epidermal
and McCoy, synthesis fibroblasts
collagen and keloid 341-346.
B.J. of in
Growth
(1979) normal vitro.
effects T. (1982) Differential acid on the growth of normal fibroblast" in vitro" from human, swine and rabbit Biol. Inter. Reps. 6: 163-170.
and Burgoon,
Bescanceney,
ultrastructure 3:
A. and Savolainen,
J.
expression in keloids: Analysis of gene metabolism and Type 1,111, IV and V prokeloid mRNAs in keloid tissue and J. Invest. Dermatol. 89: cultures.
Carpenter,
Harper,
and analysis of fibroblast cultures.
of
C.D.
a keloid.
and Odum, R.B. J.
Amer.
Acad.
(1980) The Dermatol.
50-57.
Kumegawa, M., Hatakeyama,
Yajima, T., Hiramatsu, K. and Namba, M. (1981)
stimulates Factor derived epithelial Acta 675: 305-308.
collagen clone cells.
M.,
Ikeda,
E.,
Epidermal Growth liversynthesis in Biochem. Biophys.
Cell Biology
Lembach, extracellular stimulation Epidermal
International
K.J.
(1976) accumulation of quiescent, Growth Factor.
Reports,
Vol. 73, No. 4, April
7989
synthesis Enhanced and of hyaluronic acid during human fibroblasts by mouse J. Cell. Physiol. 89:
277-288.
Lever, W.F. and SchaumburgLever, G. tissue. In: Histopathology fibrous Lippincott, Philadelphia. 577-578, Moriarity, Epidermal cells in 203:
(1983) Tumours of of the skin. pp.
Interaction of D.M. and Savage, C.R. (1980) Growth Factor with adult rat parenchymal primary culture. Arch. Biochem. Biophys.
505-518.
Murray, Keloids:
J-C., a
Pollack, review.
and Pinnell, S.V. J. Amer. Acad.
S.R. (1981) Dermatol. 4:
461-470.
Russell, J-D., Russell, effect of histamine fibroblasts isolated Physiol. 93: J. Cell.
S.B.
and Trupin, K.M. (1977) The on the growth of cultured from normal and keloid tissue. 389-394.
Russell, J.D. and Trupin, Russell, S-B., Alteration of amino acid transport cortisone: different effects in human derived from normal skin and keloid. J. 257: 9525- 9531. Russell, S-R., Trupin, K-M., Russell, J.D. and Trupin, growth-factor requirement fibroblasts for may account 85: 587-591. Nat. Acad. Sci. Savage, C.R. Factor and 7609-7611.
and Cohen, S. a new derivative.
J.S. (1982) by hydrofibroblasts Biol. Chem.
Rodriguez-Eaton, S., J.S. (1988) Reduced keloid-derived of growth. tumour Proc. (1972) J.
Epidermal Growth Biol. Chem. 247:
R.A. (1981) Human Epidermal Savage, C.R. and Harper, Growth Factor/ Urogastrone: Rapid purification procedure partial characterisation. and Analyt. Biochem. 111: 195-202. Scatchard, G. (1949) The attractions of proteins small molecules and ions. Ann. New York Acad. 51: 660-675.
for Sci.
Cell Biology
International
Reports,
Vol. 13, No. 4, April
335
1989
Schaudies, R-P., Harper, R.A. and Savage, C.R. (1985) 1251-EGF binding to responsive and non-responsive cells in culture: LOSS of cell associated radioactivity relates to growth induction. J. Cell Physiol. 124: 493-498. (1980) Tumours of Smith, J.L. (eds.). Helwig and F.K. Mostofi Williams and Wilkins, Baltimore.
Paper
received
12.5.88
the Corium. In: E.B. The skin. pp 553-557,
Paper
accepted
12.12.88