Quantitative assay of types I and III collagen synthesized by keloid biopsies and fibroblasts

384

Biochimica et Biophysica Acta, 586 (1979) 384--390 © Elsevie~/North-Holland Biomedical Press

BBA 28989

QUANTITATIVE ASSAY O F TYPES I AND III COLLAGEN SYNTHESIZED BY KELOID BIOPSIES AND FIBROBLASTS

JOHN N. CLORE, I. KELMAN COHEN and ROBERT F. DIEGELMANN

Department o f Surgery, Division o f Plastic and Reconstructive Surgery, Medical College of Virgin&, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.) (Received December 19th, 1978)

Key words: Collagen deposition; Keloid; Type I collagen; Type Ill collagen; Wound healing; (Human tissue)

Summary Molecular sieve column chromatography was used to determine the amount of t y p e I and III collagen synthesized b y normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized b y fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts. However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.

Introduction Keloids are raised, healed wounds which extend b e y o n d the original wound margins, often recur after excision and are found only in man [1]. Histologically, keloid is characterized by an apparent overabundant deposition of collagen. Studies of collagen metabolism in keloid have partially explained these clinical and histological observations because collagen synthesis by keloid biopsies [2--4] and cultured fibroblasts derived from these lesions [5] is greater than normal skin or scar. Abbreviation: Tricine, N-Tris(hydroxymethyl)methylglycine.

385 The types of collagen found in keloid were studied for several reasons. Firstly, because keloid is similar to an early healing skin w o u n d with respect to increased collagen synthesis [2--5], elevated water content [3], increased soluble collagen [6] and increased histamine content [7,8], we reasoned that keloids may have increased t y p e III collagen as has been postulated for early wounds [9,10]. Secondly, because an alteration in the relative amounts of the collagen types I and III has been observed in other disease states [11--14], one might expect that the ratio of t y p e s I and III in keloid would be altered compared to normal skin. In addition, we reasoned that there may be an increase in t y p e I collagen, which is more resistant to proteolysis than t y p e III [15] and which might explain the increased collagen deposition in keloid. Materials and Methods

Study population. Sterile surgical specimens of human keloid and normal skin were obtained from subjects {15--60 years old) within 30 rain after removal. The mean age of the normal subjects was 36 years and of the keloid patients, 25 years. All of the keloid patients and 54% of the normal subjects were black and 62% of the normal subjects and 83% of the keloid patients were female. Fibroblast isolation and culture conditions. Epidermis and subdermal fat were removed from biopsies of normal skin and keloid. The specimens were minced '(1--2 mm 3) in sterile tissue culture plates (100 mm) and overlaid with 5 ml of Eagle's medium (Gibco, Grand Island, NY) supplemented with 10% fetal calf serum, penicillin, streptomycin and 25 mM Tricine buffer (pH 7.4). The cultures were incubated at 37°C under normal atmosphere for 10--14 days, and refed every third day. After 12--14 days, the fibroblasts were harvested from the primary cultures by treatment with 0.25% trypsin and replated. Fibroblasts were the only cell t y p e present, as confirmed b y gross morphology and electron microscopy. Conditions for incubation of chick embryo calvaria, human biopsies and fibroblasts with radioactive proline. Calvaria were removed from sixteen, 15day-old chick embryos and incubated for 2 h at 37°C in 10 ml Krebs-Ringer medium containing 0.1 mM ascorbate, /3-aminopropionitrile (0.5 mM, Aldrich Chemical Co., Metuchen, NJ) and 50 t~Ci of [~4C]proline (240 Ci/M, New England Nuclear). fi-Aminopropionitrile was added to prevent collagen crosslink formation. To prepare human biopsy specimens, 400 mg tissue were minced and incubated in 12 ml Krebs-Ringer medium with fi-aminopropionitrile (0.5 mM), and 40 pCi [ 5 ) H ] p r o l i n e (49 Ci/mM, Schwanz-Mann, Orangeburg, NY) for 2 h. To prepare collagen from fibroblast cultures, confluent plates of fibroblasts in the fourth passage were incubated in Eagle's medium without serum and supplemented with 0.01 mM ascorbate, 0.5 mM ~-aminopropionitrile and [3H]proline (2 pCi/ml). After 6 h incubation, the medium and cells were removed, the plates rinsed with 1 vol. cold deionized water, and 10--20 mg uterine l e i o m y o m a collagen, containing known amounts of types I and III collagen, was added as a non-radioactive carrier. Collagen preparation from biopsies and fibroblasts. Homogenates obtained

386 from either chick calvaria, fresh human biopsies or fibroblast cultures were suspended in 0.5 M acetic acid, pepsin was added to a final concentration of 1 : 40 (w,/w) and the samples stirred at 4°C for 72 h. Collagen solubilization by pepsin was carried o u t at 4°C to minimize any selective digestion of t y p e III collagen [16,17]. Following addition of NaC1 to 12%, the precipitate was resuspended in 0.5 M acetic acid and dialyzed against 0.02 ionic strength phosphate buffer (pH 7.4). The resulting precipitate was then dialyzed against 0.1 M acetic acid and dried b y lyophilization. Separation o f collagen types I and III by molecular seive chromatography. The lyophilized samples were dissolved in 1 M CaC12/0.05 M Tris-HC1 (pH 6.5) and warmed to 55°C for 15 min. The solubilized collagen preparation was chromatographed on a 1.5 × 155 cm column of Bio-Gel agarose A-5 M (200--400 mesh, Bio-Rad, Richmond, CA) equilibrated with 1 M CaC12/0.05 M Tris-HC1 (pH 6.5) at room temperature (flow rate, 10 ml/h [18]). 2.5-ml fractions were monitored for collagen at 230 nm. Aliquots were dissolved in 10 ml Biocount (Research Products International Corp., Elk Grove Village, IL) and counted with a liquid scintillation spectrometer. Radioactivity in region A ( ~ p e III collagen) was divided b y the sum of the radioactivity in regions A--C × 100 to determine the percentage o f t y p e III collagen (Fig. 1). Identification o f type III collagen in region A. Normal dermal fibroblasts were prepared as described above and incubated in Eagle's medium without serum and supplemented with 0.3 mM ascorbate, 0.5 mM ~-aminopropionitrile and 20 gCi/ml [3SS]cystine (447.9 Ci/mM, New England Nuclear, Boston, MA) for 24 h at 37°C. The collagen was then prepared as described above and collagen types I "and III were separated by column chromatography using agarose A-5 M gel beads. In another experiment, uterine leiomyoma collagen containing a mixture of collagen types I and III was reduced with ~-mercaptoethanol and then alkylated with iodoacetic acid [19]. The sample was dialyzed against deionized water, lyophilized and chromatographed on the agarose A-5 M column as described above. In a further study, the void volume (Fig. 1, region A) obtained from chromatography on the agarose A-5 M column was dialyzed against deionized water, lyophilized, and reduced and alkylated as above. The resulting sample was then hydrolyzed in 6 N HC1 for 18 h and the presence of S-carboxymethyl cysteine was determined with a Beckman 19C amino acid analyzer. Statistical analysis. Student's t-test for independent variables was used for statistical analyses. Results

Resolution o f types I and III collagen by A-5 M column chromatography Molecular sieve column chromatography using agarose A-5 M gel beads separated the pepsin-solubilized, radioactive collagen into three regions (Fig. 1 ). Region A contained trimers of t y p e III collagen, region C contained a~ and a2 monomers of t y p e I collagen and region B contained cross-linked dimers of t y p e I collagen [18]. These conclusions were based on the following experiments. Reduction and alkylation of region A resulted in a product which no

387

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F i g . 1. Elution profile o f pepsinoSoinbflized collagen chromatographed on a 1 . 5 X 1 5 5 c m a g a r o s e A - 5 M column. • ~ , absorbance at 2 3 0 n m of purified uterine l e i o m y o m a carrier collagen; o o elution profile o f [3H]proline collagen. Region A, type III collagen trimers; region B, type I collagen d i m e r s ; region C, type I collagen m o n o m e r s .

longer chromatographed in that region, but co-chromatographed with type I a chains (region C) and amino acid analysis of the reduced and alkylated collagen in region A showed the presence of S-carboxymethyl cysteine. In addition, when normal dermal fibroblasts were incubated with [3SS]cystine, greater than 81% of the radioactive collagen eluted with type III collagen (peak A). The remaining 19% of the 3ss radioactivity was in peak C, while no detectable activity was found in region B. Conversely, A-5 M column chromatography of radioactive chick embryo calvaria collagen (type I collagen) showed that 89% of the newly synthesized collagen was in peak C, 11% in peak B and no detectable radioactivity was observed in peak A. Although ~-aminopropionitrile reduced the amount of cross-linked type I collagen in region B, this species was not eliminated completely. There was no selective loss of either type I or III collagen when regions A and C were each pooled, concentrated and re-chromatographed separately.

Quantitation of type III collagen synthesized by keloid and normal skin Using the A-5 M column to separate and quantitate types I and III collagen, TABLE

I

DISTRIBUTION

OF TYPE

III C O L L A G E N

IN K E L O I D

For percent type III the radioactivity in region A from agarose A-5 M column chromatography (Fig. 1) was divided by total radioactivity in regions A--C × 100. Specimen

N u m b e r of observations

% type III (+ S.E.)

Normal dermis Keloid biopsy N o r m a l dermis fibroblasts Keloid fibroblasts

5 6 13 12

19.6 21.9 5.7 6.5

-+ 2.7 +_ 1.9 -+ 0.8 +- 0 . 8

388 we found the percentage of t y p e III collagen synthesized b y fresh biopsies of keloids from six patients was not significantly different than five normal skin controls (Table I). Likewise, no significant differences were observed in the percentage o f ' t y p e III collagen of twelve keloid fibroblast cultures compared to 13 normal skin fibroblast cultures (Table I). Both normal dermis and keloid fibroblasts had a significantly reduced percent of t y p e III collagen compared to fresh biopsies (Table I). Biopsies of keloids from t w o patients treated with intralesional triamcinolone contained 23.8% and 19.9% t y p e III collagen. Discussion

Normal dermis contains t w o distinct collagen types. Type I collagen is composed of t w o ~1 (I) chains and a single ~2 chain in a triple helix. Type III collagen consists of three identical ~1 (III) chains with intramolecular sulfhydryl bonds in the helical region which are not found in t y p e I collagen [16]. Type III collagen constitutes approximately 20% of normal adult dermis .[20], b u t this percentage may vary in certain normal physiologic and disease processes. For example, normally t y p e III collagen is the predominant form in the fetus, b u t the ratio of t y p e I:III increases sharply b y the time of birth [20]. Examples o f disease states characterized by an alteration of types I and III collagen include atherosclerotic plaques, which contain significantly less t y p e III collagen and more t y p e I than normal vessel walls [12] and t y p e IV Ehler'sDanlos syndrome where t y p e III collagen is absent [11]. Conversely, osteogenesis imperfecta is characterized by decreased t y p e I collagen in bone and skin fibroblasts [ 13 ]. Similarly, there have been two preliminary reports suggesting increased type III in healing skin wounds. Bailey et al. [9] found increased t y p e III collagen in both acute and chronic rat skin wounds. However, turpentine was used to cause the acute response resulting in a highly vascularized granuloma and polyvinyl sponge implants were used for the chronic foreign b o d y wounds. These wounds are not normal wounds and are characterized b y increased vascularity which may explain the observed increase in t y p e III collagen. In another study, Bailey et al. [21] found 'slightly higher' t y p e III collagen in hypertrophic scars compared to normal human dermis. However, this conclusion may also be inaccurate because quantitation was based on the weights of precipitates of collagen obtained b y salt fractionation rather than more precise methods of quantitation. Gay et al. [10] studied human skin w o u n d material collected in a subcutaneous cellstic implant. Using immunofluorescent staining, they detected increased t y p e III collagen up to 72 h post-wounding. Once again, one must question the significance of observations made using a foreign b o d y material. The chromatographic procedure used in the present studies allows separation and quantitation of types I and III collagen. This method utilizes the presence of disulfide bonds in the helical region of type III collagen. The disulfide bridges stablize the triple helical (type III) molecule as a 300 000
389 A-5 M column chromatography (Fig. 1) has been well established by others [18,22] as well as by the present studies. Furthermore, there was no evidence of type I ~ chains in region A when chick embryo calvaria type I collagen was synthesized under the incubation conditions used in these studies and analyzed by agarose A-5 M column chromatography. The presence of 3sS radioactivity in region C following chromatography of [3SS]cysteine-labeled collagen may represent a small amount of type I procollagen remaining after pepsin treatment [17]. Although, the actual content of type III collagen reported in these studies may be underestimated due to pepsin digestion [17], it is unlikely that there would be any selective loss of type III collagen from the keloid specimens compared to the normal skin controls. The present findings demonstrate that there is no significant difference in the ratio of types I and III collagen produced by fresh biopsies and fibroblasts from keloid when compared to normal human skin controls (Table I). However, there was a greater percentage of type III collagen synthesized by fresh biopsies of both normal dermis and keloid when compared to cultured fibro. blasts derived from these sources. This may be due to the presence of type IIIproducing endothelial cells in fresh biopsies which are no longer present in the fibroblast cultures. Alternatively, the de~lifferentiation of fibroblasts following four cell passages may alter the types of collagen produced. A similar observation was made by Mayne et al. [23] who demonstrated alterations in the synthesis of collagen types by cultured chondrocytes after multiple passages. Because intralesional triamcinolone causes keloid resorption [24], it was hypothesized that keloid regression may be accompanied by an alteration of collagen types. However, there was no significant difference in the percent type III collagen synthesized in keloids from two patients following intralesional triamcinolone treatment. Possibly, the distribution of collagen types I and III is altered during the initiation of keloid formation but early specimens cannot be obtained for such a study. We conclude from these studies that an alteration in the ratio of collagen types is not related to the increased collagen synthesis characteristic of keloids. Acknowledgments This investigation was supported by Grant Number GM-20298, awarded by the National Institutes of Health. The authors are grateful for the fine technical assistance of Ms. Mary Anna Link and Ms. Benita Atiyeh and the typing assistance of Ms. Rae Spivey and Ms. Candace Reed. References 1 Peacock, E.E., Jr., Madden, J.W. and Trier, W.C. (1970) South. Med. J. 6 3 , 7 5 5 - - 7 6 0 2 Cohen, I.K., Keiser, H.R. and Sjocrdsma, A. (1971) Surg. F o r u m 2 2 , 4 8 8 - - 4 8 9 3 Cohen, I.K., Diegelman, R.F. and Keiscr, H.R. (1976) in The Ultrastructure of Collagen (Longacre, J.J., ed.), pp. 199--212, Charles C. Thomas, Springfield, IL 4 Craig, R.D.P., Scholfield, J.D. and Jackson, S.S. (1975) Eur. J. Clin. Invest. 5, 69--74 5 Diegclman, R.F., Cohen, I.K. and McCoy, B.J. (1979) J. Cell Physiol. 98, 341--346 6 Harris, E.D. and Sjoerdsma, A. (1966) Lancet 2 , 7 0 7 - - 7 1 1 7 Cohen, I.K., Beaven, M.A., Horakova, S. and Keiser, H.R. (1972) Surg. F orum 2 3 , 5 0 9 - - 5 1 0

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