JOURNALOF

Dermatological Science

ELSEVIER Journal of Dermatological Science 12 (1996) 1633171

Localization of prolidase gene expression in scar tissue using in situ hybridization

Yasuko Senboshi, Takashi Oono, Jir6 Arata* Department of Dermatology, Okayama University Medical School, Shikata-cho 2-5-1, Okayama 700, Japan

Received 26 September 1995; revised 10 November 1995; accepted 22 November 1995

Abstract

We investigated prolidase gene expression in human skin by means of Northern blot analysis and in situ hybridization. Northern blot analysis revealed that an mRNA species that was specific for prolidase was present in cultured human skin fibroblasts and keratinocytes. In situ hybridization using non-isotopic riboprobes labeled with digoxigenin and an isotopic riboprobe labeled with [35S]UTP localized prolidase gene expression to fibroblasts and endothelial cells of small vessels in scar tissue. Prolidase mRNA was also prominently expressed in keratinocytes near the basal layer overlying scar tissue. These findings indicate that prolidase may have an important role in wound healing.

Keywords: Prolidase; Wound healing; Scar tissue; In situ hybridization

1. Introduction

Wound healing is a highly regulated event. Wound healing has been arbitrarily divided into three phases: inflammation, granulation tissue for- mation, and matrix formation and remodeling [I]. Recent experiments have shown that col- lagenolytic activity has an important role in wound healing [2,3]. Wounded tissue re-attains tensile strength not only by new collagen deposi- tion but also by collagen remodeling including the formation of large collagen bundles and an alter- ation of intermolecular cross-links. The tissue re- modeling is regulated by a balance of collagen

* Corresponding author.

synthesis and degradation. Collagen degradation is initiated by collagenase [3]. Hembry and Ehrlich [2] recently reported that immunoprecipi- tation of collagenase was found in actively remod- eling areas of scar tissue. In the epidermis, collagenase gene expression is increased in kerati- nocytes located at the edge of acute injuries and is decreased after re-epithelialization [4-61. Collage- nase immunoreactivity expression has been ob- served by means of immunohistochemistry in the areas of active resorption and in some capillaries in the dermis of hypertrophic scars [2]. This evi- dence suggests that collagenolytic activity has a role in both the acute and late phases of wound healing.

Prolidase (peptide D) is a ubiquitous enzyme that hydrolyses iminodi- and/or iminotri- peptides

0923-181 l/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDZ 0923-1811(95)00505-M

164 Y. Senboshi et al. 1 Journal of Dermatological Science 12 (1996) 163-l 71

that contain a carboxy-terminal proline. The en- zyme appears to have a role in the final step of degradation of proteins, including collagen, and in recycling proline for collagen synthesis [7,8]. Recently, complementary DNA clones of human prolidase were isolated and sequenced [9]. Se- quence analyses have shown that human erythro- cyte prolidase and human skin collagenase are evolutionarily related [lo]. Prolidase deficiency causes various dermatologic symptoms such as recalcitrant leg ulcers [11,12]. Taken together, this indicates that prolidase may be involved in the process of wound healing, however, the role of prolidase in the skin is poorly understood. In the present study, therefore, we examined prolidase gene expression in human scar tissue.

The results of the present study indicate that the prolidase gene was expressed in dermal fibrob- lasts, dermal capillaries, and the epidermis in and around both hypertrophic and normal scars, while the expression of the prolidase gene in normal skin was negligible.

2. Materials and methods

2.1. Tissue samples

A biopsy specimen for non-isotopic in situ hy- bridization was obtained from a 17-year-old male who had had a hypertrophic scar for 5 months after a skin infection. The specimen was taken under local anesthesia. Scar samples for isotopic in situ hybridization were obtained from five pa- tients (three females and two males, aged 6-80 years). The age of the scars varied from 3 weeks to 24 months. A control skin sample was obtained from a 19-year-old female who received skin surgery. The tissues were frozen in liquid nitrogen immediately after excision and stored at - 80°C until used for in situ hybridization. All skin sam- ples were obtained after informed consent had been obtained.

2.2. Cell culture

Fibroblast cultures were established by out- growth from a control skin biopsy. The cells were

maintained in Dulbecco’s modified Eagle’s medium (GIBCO BRL, Gaithersburg, MD), sup- plemented with 10% fetal calf serum (FCS), 50 pg/ml sodium ascorbate, 300 pgg/ml glutamate, 100 U/ml penicillin, 100 pgg/ml streptomycin, and grown in the moist atmosphere of a CO, incuba- tor (5% CO,, 95% air) at 37°C.

Keratinocyte (Epipack@)(Kurabou, Osaka, Japan) cultures were established using commercial outgrowth. The cells were maintained in kerati- nocyte serum-free medium @FM) (GIBCO BRL), and grown under the same conditions as the fibroblast cultures.

2.3. RNA isolation and Northern blot analysis

Monolayer cultures were lysed in 4 M guanid- ium thiocyanate containing 0.1 M 2-mercap- toethanol. DNA was sheared by pumping the lysate six times through a 22-gauge needle. RNA was then separated by ultracentrifugation at 35000 x g for 20 h at 18°C through a 5.7 M cesium chloride cushion according to standard protocol [13]. For Northern blot analysis, total RNA (10 pg/lane) was separated by gel elec- trophoresis on a 1% agarose gel under denatured conditions, transferred onto nylon membranes (hybon-N@) (Amersham, Buckinghamshire, UK) by capillary blotting. After crosslinking RNA to the filters by baking for 2 h at 80°C the filters were prehybridized in 50% formamide, 0.1% sodium dodecyl sulfate (SDS), 5 x SSC (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate), 5 x Denhardt’s solution (1 x Denhardt’s is 0.02% bovine serum albumin (BSA), 0.02% Ficoll, and 0.02% polyvinylpyrrolidone), and 100 p g/ml sonicated herring sperm DNA (GIBCO BRL). They were then hybridized in the buffer described above at 42°C overnight with the prolidase spe- cific cDNA probes radioactively labeled with [32P]dATP using a Pharmacia@ DNA labeling kit by random priming to specific activities of 2-5 x lo8 cpm/pg. After hybridization, filters were washed twice in 2 x SSC, 0.1% SDS at room temperature for 15 min, followed by washing in 0.1 x SSC, 0.1% SDS at 60°C for 15 min. Filters were then exposed to radiosensitive film at - 80°C for 6 days. An insert isolated from the

Y. Senboshi et al. / Journal of Dermatological Science 12 (1996) 163-l 71 165

plasmids pEPD-W covering the entire coding re- gion of prolidase was used as the hybridization probe.

2.4. cRNA probes

To generate a cRNA probe for the detection of prolidase mRNA species by in situ hybridization, the 1.4-kb EcoRl fragment was isolated from the original cDNA clone pEPD-W kindly provided by Dr. F. Endo (Department of Pediatrics, Ku- mamoto University Medical School, Kumamoto, Japan) and sub-cloned into a pGem 3 vector containing the SP6 and T7 RNA polymerase pro- moters on either side of a multiple cloning site (p-Koe 1). For the production of the antisense cRNA probe and the sense cRNA probe, the plasmid p-Koe 1 was linearized with PvuII and HindA, respectively. In vitro transcription was carried out with SP6 RNA polymerase for the antisense cRNA probe or with T7 RNA poly- merase for the sense probe according to the in- structions from the supplier. Plasmid Hf-677 with a 1.5-kb insert specific for the C-terminal end of the alpha 1 (I) chain 1141 was used as the probe for collagen I mRNA. For the production of the antisense cRNA probe of collagen alpha 1 (I), the plasmid was linearized Xho I. In vitro transcrip- tion was carried out with SP6 RNA polymerase for the antisense cRNA probe. Briefly, for non- isotopic in situ hybridization, 1 pug linearized plas- mid, 2 ~1 nucleotide labeling mixture (10 mmol/l ATP, 10 mmol/l CTP, 10 mmol/l GTP, 6.5 mmol/ 1 UTP and 3.5 mmol/l digoxigenin-labeled UTP in Tris-HCl, pH 7.5), 1 ~1 RN&e inhibitor, 1 ~1 dithiothreitol (DTT) (0.2 mol) and 2 ~1 SP6 or T7 RNA polymerase was added to the reaction buffer. The volume was brought to 20 ~1 with diethyl pyrocarbonate (DEPC)-treated water.

For isotopic in situ hybridization, transcription was carried out in the presence of 1 pg linearized plasmid, 1 ~1 DTT (0.2 mol), 1 ~1 RNase, 1.5 ,LLI nucleotide mixture (10 mmojl ATP, 10 mmol/l GTP, 10 mmol/l CTP), 1 ~1 cold UTP (200 PM), 4 ~1 [35S]UTP (2 mM), and 1 ~1 SP6 or T7 RNA polymerase, brought to a total volume of 20 ~1 with DEPC-treated water. Each mixture was incu- bated for 2 h at 37°C. The RNA probe was

precipitated from .0.3 M sodium acetate with ethanol and redissolved in 10 ~1 water.

2.5. In situ hybridization

In situ hybridization was carried out according to a previously described method with minor modificatioas [15]. Briefly, 5-pm frozen sections mounted on slides were washed twice in 2 x SSC followed by an acetylation step in acetic anhy- dride-triethanolamine (pH 8.0) to reduce non-spe- cific binding. The slides were then rinsed in 2 x SSC and incubated in 0.1 M Tris-HCl (pH 7.0), 0.1 M glycine for 30 min, washed again in 2 x SSC, subsequently equilibrated in 50% formamide and 2 x SSC at 50°C. Prior to hybridization, the RNA probes were denatured in 5 ~1 50% for- mamide, 200 mM DTT in 2 x SSC, 1 ~1 herring sperm DNA, 1 ~1 tRNA, and 1 ~1 BSA at 95°C for 5 min. Hybridization was performed at 42°C overnight. The activity of the cRNA .probe for isotopic in situ hybridization was 1 x lo6 counts/ min/section. After hybridization, the slides were rinsed in 50% formamide and 2 x SSC at 60°C followed by six washes in 2 x SSC. After treat- ment of the section with RNase A in 2 x SSC at 37°C for 30 min, the slides were washed in 50% formamide, 2 x SSC at 60°C for 30 min, then rinsed in Z! x SSC, and finally dehydrated in a graduated ethanol series. For isotopic in situ hy- bridization, autoradiography was performed using autoradiography emulsion (Kodak) diluted 1: 1 with water and adjusted to 37°C. After exposure of the slides for 2 or 3 weeks at 4°C in light-proof boxes with fresh silica gel, the slides were devel- oped, fixed, and finally stained with hematoxylin- eosin or methyl green.

2.6. Digoxigenin detection step for non-isotopic in situ hybridization

The condition for non-isotopic in situ hy- bridization is identical to that of isotopic in situ hybridization which was described above. All pro- cedures were carried out at room temperature. Before detection, the slides were rinsed in phos- phate-buffered saline (PBS) for 5 min. The slides were incubated for 20 min with 0.6% hydrogen

166 Y. Senboshi et al. / Journal of Dermatoiogical Science 12 (1996) 163- 171

peroxide in methanol to quench endogenous per- oxidase activity. After washing in PBS for 5 min, the slides were incubated with 3% FCS in PBS for 15 min. Then the slides were incubated with horse-radish peroxidase (HRP)-conjugated sheep anti digoxigenin antibody (1:30) for 60 min in a moist chamber. The slides were washed twice in PBS for 5 min to remove unbound conjugated antibody. Control experiments were performed using PBS instead of HRP-conjugated antibody. The color reaction was produced using 3,3,-di- aminobenzidine tetrahydrochloride (DAB) at a final concentration of 0.06% for 30 min. The slides were washed in water, air dried, and stained with hematoxylin.

3. Results

3.1. Northern blot analysis

In order to confirm the presence of mRNA specific for prolidase, total RNA was isolated from cultured fibroblasts and keratinocytes. After electrophoresis in 1% agarose, RNAs were trans- ferred to nylon membranes and hybridized to the [32P]ATP-labeled cDNA probe specific for proli- dase. A single band (2.3 kb) was observed, indi- cating the presence of an mRNA species that was specific for prolidase in human skin fibroblasts and keratinocytes (Fig. 1).

3.2. Non-isotopic in situ hybridization

Recent experiments using in situ hybridization have indicated that the expression of the collagen alpha 1 (I) gene is upregulated in scar tissue [16]. Therefore, in situ hybridization using the collagen alpha 1 (I) antisense cRNA probe was performed as a positive control. As expected, the expression of the collagen alpha 1 (I) gene was observed in the spindle-shaped fibroblast-like cells in the der- mis of 5-month-old hypertrophic scar tissue, indi- cating the presence of intact message and the integrity of the methods (Fig. 2). When the proli- dase antisense cRNA probe was applied, positive staining indicating the expression of the prolidase gene was observed in the fibroblast-like cells and

endothelial cells of small vessels in the dermis (Figs. 3 and 4). Only background staining was observed when the prolidase sense cRNA probe was applied (Figs. 3 and 4). Prolidase gene expres- sion was also observed in the epidermal cells in the lesion of the scar (Fig. 5). When the prolidase antisense cRNA probe was applied to sections of control skin, only background staining was ob- served (data not shown).

3.3. Isotopic in situ hybridization

The results of non-isotopic in situ hybridization suggest that the expression of the prolidase gene is upregulated in hypertrophic scar tissue. So far the localization of prolidase gene has not been re- ported. To confirm our result obtained by non- isotopic in situ hybridization, we performed isotopic in situ hybridization using chronologi- cally different scar tissues (aged 3 weeks to 2 years). A total of five scar tissues were subjected to in situ hybridization. Distinct labeling was

Northern blot -28s

1 i Ii

2.3 kb

Keratinocyte Fibroblast Fig. 1. A single band (2.3 kb) was observed in human fibrob- lasts and keratinocytes.

Fig. 2 using

Fig. 3

stainir

Y. Senboshi et al. /Journal of Dermatological Science 12 (1996) 163-l 71 167

The non-i,

Non led in ig on1

exp1 sot01

-isot I the lY.

‘es ?ic

OP fil

iion in 5

ic in srot

of iitu

1 sil >lar

collagen alpha 1 (I) gene was observed in the fibroblast-like cells in the dermis L hybridization.

of 5-l month- .old scar t

tu hybridization in scar tissue. (A) Prolidase antisense cRN.4 probe was applied The positi\ fe st aining was it-like cells of the dermis. (B) Application of the prolidase sense cRNA pro1 3e res :ulted i In b ackgrc lurid

168 Y. Senboshi et al. /Journal of Dermatological Science 12 (1996) 163-l 71

Fig. 6 The endothelial cells of small vessels (open arrow) and fibroblast (solid arrow) were hybridized with prolidase ar probe using isotopic in situ hybridization.

Fig. I Prolidase gene was also observed in the epidermis.

detec :ted in all scar tissues. Prolidase gene expression was c observed in fibroblast-like cells in the dermis and endo lthelial cells of small vessels in all scar tissues (Fig. 6). There was no evidence of increased proli- dase gene expression in the papillary dermis. Proli-

Y. Senboshi et al. /Journal of Dermatological Science 12 (1996) 163-l 71 169

iti-s

dase gene expression was also observed in the : 01 lying epidermis (Fig. 7). The signals for prc Aid gene were observed in the entire epidermis. Hc ever, the intensity of labeling was stronger in basal layer than that of the suprabasal layer (1 Fig

:nse

rer-

3w- the .7).

170 Y. Senboshi et al. / Journal oj’Dermatologica1 Science 12 (1996) 163- 171

4. Discussion

The present study shows that the prolidase gene was expressed in scar tissue. The distribution pat- tern, however, is different from that previously reported for collagenase [2]. Namely, prolidase gene expression was observed in all fibroblast-like cells and endothelial cells of small vessels in the dermis. In contrast, collagenase immunoprecipita- tion is primarily located in areas of active resorp- tion. Moreover, prolidase gene was observed in the epidermis. No collagenase gene has been ob- served in the epidermis of scar tissue [2]. The fact that collagenase and prolidase have different dis- tributions may indicate that the gene expression of these enzymes is regulated by different mecha- nisms in scar tissue. Transforming growth factor- p is thought to modulate collagenase gene expression [17], however, cytokines that regulate prolidase gene expression have not yet been de- scribed.

Several authors have shown that prolidase ac- tivity is increased for a few weeks after injury in rodent skin [l&19]. Data from the present study indicates that at the mRNA level, prolidase gene expression was to some extent upregulated in relatively old scar tissue compared to normal skin. The collagenase immunoprecipitate was increased in areas of active remodeling. These data are consistent with the hypothesis that prolidase hy- drolyzes imidodipeptide derived from collagen de- graded by collagenase in the remodeling process. There may be some prolidase gene expression in normal skin, but at such low levels as to be undetectable in the present study.

We observed prolidase gene expression in en- dothelial cells and the epidermis. Recent studies have shown that collagenase gene expression is upregulated in wound-edge keratinocytes and is down-regulated again after re-epithelialization [4- 6]. At the early phase of wound edge, the signals for collagenase were observed in basal kerati- nocytes [6]. The disruption of the basement mem- brane and the exposure of the keratinocytes to interstital collagen were thought to stimulate col- lagenase production [5,6]. In our data, the signals for prolidase in the basal layer localized by iso-

topic in situ hybridization is stronger than that of the suprabasal layer. This finding may indicate that basal keratinocytes are mainly involved in producing prolidase gene. The presence of proli- dase in the epidermis of scar tissue after re-epithe- lialization suggests that this enzyme is necessary after re-epithelialization. In prolidase-deficient pa- tients, various skin manifestations such as recalci- trant leg ulcers, hyperkeratotic lesions [20], and ichthyosis have been reported. Thinner epidermis consisting of only two or three layers of squamous-cells has also been reported in this dis- ease [21]. These observations may indicate that this enzyme is important in maintaining the nor- mal architecture of the epidermis.

The role of prolidase in endothelial cells of small vessels is presently unclear. Thickened der- ma1 vessel basement membrane [22-241 and vas- cular occlusion in acute ulceration [25,26] have been observed in prolidase deficiency using light microscopy. Amyloid fibril-like deposits around dermal capillaries [20], lamellated and interrupted basal lamina around dermal small vessels and amyloid deposition [21], and multilaminated basal lamina around the dermal vessels [27] have been observed in prolidase deficiency using electronmi- croscopy: The relationship between altered colla- gen metabolism and prolidase remains unclear, although some authors speculate that these changes may reflect a disturbance in connective tissue metabolism due to a deficiency in prolidase [27]. The existence of prolidase gene expression in the small vessels of scar tissue is consistent with this hypothesis.

To elucidate the mechanism of prolidase gene expression, the role of cytokines such as trans- forming growth factor-p in the gene expression of this enzyme should be investigated.

Acknowledgements

We would like to thank Dr. F. Endo for kindly providing the original cDNA clone pEPD-W.

References

[l] Richard AF, Clark MD, Denver CO: Cutaneous tissue

Y. Senboshi et al. /Journal of Dermatological Science 12 (1996) 163-171 171

repair: Basic biologic consideration. Am Acad Dermatol 13: 701-725, 1985.

[2] Hembry RM, Ehrlich HP: lmmunolocalization of colla- genase and tissue inhibitor of metalloproteinases (TIMP) in hypertrophic scar tissue. Br J Dermatol 115: 409-420, 1986.

[3] Clark RAF: Mechanisms of cutaneous wound repair. In Dermatology in General Medicine, 4th edition. Edited by TB Fitzpatrick, AZ Eisen, K Wolff, IM Freedbelg, KF Ansten. McGraw-Hill, New York, 1993, pp. 473-486.

[4] Saarialho-Kere UK, Chang ES, Welgus HG, Parks WC: Distinct localization of collagenase and tissue inhibitor of metalloproteinases expression in wound healing asso- ciated with ulcerative pyogenic granuloma. J Clin Invest 90: 1952-1957, 1992.

[5] Saarialho-Kere UK, Kovacs SO, Pentland AP, Olerud JE, Welgus HG, Parks WC: Cell-matrix interactions modulate interstitial collagenase expression by human keratinocytes involved in wound healing. J Clin Invest 92: 2858-2866, 1993.

[6] Inoue M, Kratz G, Haegerstrand A, Stable-Backdahl M: Collagenase expression is rapidly induced in wound-edge keratinocytes after acute injury in human skin, persists during healing, and stops at re-epithelialization. J Invest Dermatol 104: 4799483, 1995.

[7] Endo F, Matsuda I: Molecular basis of prolidase (pep- tide D) deficiency. Mol Biol Med 8: 117-127, 1991.

[8] Jockson SH, Heininger JA: Proline recycling during collagen metabolism as determined by concurrent ‘*02 and 3H-labeling. Biochim Biophys Acta 381: 359-367, 1974.

[l l] Goodman SI, Solomons CC, Muschenheim F, McIntyre CA, Miles B, O’Brien D: A syndrome resembling lath- yrism associated with iminodipeptiduria. Am J Med 45: 152-159, 1968.

[9] Endo F, Tanoue A, Nakai H, Hata A, Indo Y, Titani K, Matsuda: Primary structure and gene localization of human prolidase. J Biol Chem 264: 447664481, 1989.

[lo] Mock WL, Zhuang H: Chemical modification locates guanidinyl and carboxylate groups within the active site of prolidase. Biochem Biophys Res Commun 180: 401~ 406, 1991.

[12] Arata J, Umemura S, Yamamoto Y, Hagiyama M, Nohara N: Prolidase deficiency - its dermatological manifestations and some additional biochemical studies. Arch Dermatol 115: 62-67, 1979.

[13] Maniatis T, Fritsch EF, Sambrook J: Isolation of total RNA from mammalian cells. In Molecular Cloning, A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory, New York, 1989, pp. 7.6-7.9.

[14] Chu ML, Myers JC, Bernard MP, Ding JF, Ramirez F: Cloning and characterization of five overlapping cDNAs specific for the human pro G( 1 (I) chain. Nucleic Acid

Res 10: 5925-5934, 1982. [15] Oono T, Specks U, Eckes B, Mejewski S, Hunzelmann

N, Timple R, Krieg T: Expression of type VI collagen mRNA. during wound healing. J Invest Dermatol 100: 329-334, 1993.

[16] ScharlIetter K, Kulozik M, Stolz W, Lankat-Buttgereit B, Hatamochi A, Sohnchen R, Krieg T: Localization of collagen a 1 (I) gene expression during wound healing by in situ hybridization. J Invest Dermatol 93: 405-412, 1989.

[17] Overall CM, Wrana JL, Sodek J: Independent regulation of collagenase, 720-kDa progelatinase, and metalloendo- proteinase inhibitor expression in human fibroblasts by transforming growth factor-p. J Biol Chem 264: 1860- 1869, 1989.

[18] Umemura S, Arata J, Nohara N: Prolidase activity during healing of skin burns in rats. J Dermatol 7: 217-219, 1980.

[19] Oono ‘T, Arata J: Characteristics of prolidase and proli- nase in prolidase-deficient patients with some prelimi- nary studies of their role in skin. J Dermatol 15: 2122219, 1988.

[20] Ogata A, Tanaka S, Tomoda T, Murayama E, Endo F, Kikuchi I: Autosomal recessive prolidase deficiency: three patients with recalcitrant leg ulcers. Arch Dermatol 117: 689-694, 1981.

[21] Sekiya M, Ohnishi Y, Kimura K: An autopsy case of prolidase deficiency. Virchows Arch A Path01 Anat His- to1 406: 125-131, 1985.

[22] Leoni A, Cetta G, Tenni R, Pasquali-Ronchetti I, Bertolmi F, Guerra D, Dyne K, Castellani A: Prolidase deficiency in two siblings with chronic leg ulcerations. Clinical, biochemical, and morphologic aspects. Arch Dermatol 123: 4933499, 1987.

[23] Pasquali-Ronchetti 1, Quaglino D Jr, Dyne K, Zanaboni G, Cetta G: Ultrastructural studies on dermis from prolidase deficient subjects. J Submicrosc Cytol Path01 23: 439-445, 1991.

[24] Quaglino D Jr, Dyne K, Zanaboni G, Cetta G, Pasquali- Ronchetti I: Dermal alterations in patients affected by prolidase deficiency. Proc Fed Eur Connect Tissue Sot 12: 132, 1990.

[25] Lambert D, Larregue M, Godard W: Ulceres de jambe apparus a la puberte vraisemblablement consecutifs a un deficit en prolidase. Ann Dermatol Venereol 109: 681- 683, 1982.

[26] Pierard GE, Cornil F, Lapiere CM: Pathogenesis of ulcerations in deficiency of prolidase: the role of angio- pathy and deposits of amyloid. Am J Dermatopathol 6: 491-497, 1984.

[27] Arata J, Tada J, Yamada T, Oono T, Yasutomi H: Angiopathic pathogenesis of clinical manifestation in prolidase deficiency. Arch Dermatol 127: 124-125, 1991.