- Email: [email protected]
The relationship between quantitative changes in collagen and formation of wrinkles on hairless mouse skin after chronic UV irradiation
JOU R NA L OF
Dermatological Science
ELSEVIER
Journal of Dermatological
Science I2 (1996) 56663
The relationship between quantitative changes in collagen and formation of wrinkles on hairless mouse skin after chronic UV irradiation Yoshinori Takema, Michihiro Hattori*, Katsunori Aizawa’ Biological Sciencr Laboratories.
Tochigi, Kao Corporation
Received 26 April
2606 Akabane, Idikai,
Haga, Tochigi, 321-34 Japan
1995; revision received 2 June 1995; accepted 9 June 1995
Abstract Female albino hairless mice were irradiated chronically with sub-erythemal doses of UVB radiation. Collagen extracted from the irradiated or non-irradiated dorsal skin of mice was fractionated into neutral salt-soluble (NSC), acid-soluble (ASC) and insoluble fractions (ISC). An age-related exponential decrease in the content and proportion of acid-soluble collagen was found in each group. The contents and the proportions of ASC from irradiated mice were always significantly lower than those from age-matched control animals. Age-related slight decreases were observed in the contents (per fresh weight of tissues) of NSC, ISC and total collagen in the control group but decreases in these collagen contents after UVB irradiation were marked. A dramatic decrease in ASC occurred nearly concomitantly with wrinkle formation in the irradiated mice. The decrease of acid-soluble skin collagen in irradiated mice may play a role in the formation of wrinkles on hairless mouse dorsal skin. Kqwwdss: Acid-soluble
collagen; Wrinkle formation;
UVB;
1. Introduction Chronic UV exposure of human skin leads to alterations in the dermal connective tissue such as changes in relative composition and in deposition Ahbrr~icrtions: UVB: ultraviolet radiation in the wavelength range 290-320 nm, ASC: 0.5 M acetic acid-soluble collagen, ISC: insoluble collagen, NSC: neutral salt-soluble collagen, SDS: sodium dodecyl sulfate. * Corresponding author, Tel.: 285 68 7585: Fax: 285 68
7452. ’ Present address: National Nishisatonaka. Ohoteramachi,
Institute for Basic Biology, 38 Okasaki-City, Aichi. Japan
Hairless
pattern of the dermal matrix proteins collagen and elastin [l-4]. A number of investigators have reported successin the development of histological animal models for solar-aged skin [5-61. Of these animal models, hairless mice have been used extensively in studies of the effects of chronic UV irradiation [7-91. Some biochemical analyses of UV-exposed mouse skin have also been reported [5,8-lo]. Wrinkles develop on these mice in response to irradiation and are considered as indicators of chronic UV exposure or photo-aging [ 11- 151.Despite several reports concerning collagen metabolism in wrinkle effacement by retinoic
0923-1811~‘96~$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0923- I8 I 1(95)00467-7
mice
acid using this mouse model [16] there have been few studies of the relationship between the progressive changes in collagen and the formation of UVB-induced wrinkles in the skin in this model except on the total collagen and pepsin-soluble collagen contents [9,15]. Chatterjee et al. [9] reported that there was a relationship between the UVB-induced increase in the thickness of hairless mouse skin and the formation of wrinkles. On the other hand, Moloney et al. [IS] investigated the quantitative and qualitative changes in dermal collagen and elastin which occur in response to chronic ultraviolet irradiation and reported that sufficient UVB irradiation will eventually cause changes in dermal elastin and collagen content; wrinkle formation precedes such however, changes. Therefore, the present study was undertaken to investigate in more detail whether UVB irradiation changes the contents of skin collagen and is correlated with the formation of wrinkles on hairless mice. 2. Materials
and methods
2.1. Animds Female hairless HR/ICR mice were used in this study. This strain was established from a cross between a hairless strain, HR/HR (originally from Nisseiken Corporation, Japan), and a normal haired strain, HaM/ICR, and has been maintained in our laboratory by hairless brothernorma1 haired sister mating for several years. The animals were fed ad libitum and housed under conventional conditions at a controlled temperature (23 f 2°C) humidity (55 + 10%) and light (12 h light/l2 h darkness, without any ultraviolet emission). Animals were cared for in accordance with our institutional guidelines. 2.2. Rudiation sowce The hairless mice were separated into 2 groups (n = 15) at 4-weeks-old. One group was irradiated with UVB and the other served as a non-irradiated control group. Toshiba FL20SE lamps were used as a UVB source without any filtering. The distance from the lamps to the animals’ back was 35 cm. The irradiation energy was 0.42 mW/cm’ which was monitored with a UV-radiometer-305/
365D (Topcon Co. Ltd., Tokyo) at 305 nm (290320 nm) and MSR7000 radiospectrometer (Optical Science Co. Ltd.). Fig. 1 shows the spectral output of the UVB source. The mice were exposed 3 times each experimental week at a dose of 61 mJ&m’ (just below the erythemal dose) for 24 weeks yielding a total dose of 4.39 J/cm”. Two to five mice were housed per cage, and during exposure the animals could move around freely in the cages. At intervals of approximately 4 weeks after initiation of treatment, 2 or 3 animals in each group were killed and dorsal skin was excised for analysis. 2.3. Wrinkle nwusurenwnt Dorsal skin wrinkling caused by chronological suberythemal dose UVB exposure was graded each week according the method described by Bissett et al. [l l] as follows: grade 0, no coarse wrinkles; grade 1, a few shallow coarse wrinkles: grade 2, some coarse wrinkles; grade 3, several deep coarse wrinkles. The scale range from 0 for the normal animals to 3 for heavily wrinkled skin. 2.4. Step~ise extraction of collagen Animals were sacrificed by an overdose of chloroform, and the dorsal skin (1.57 cm’) was immediately collected. Five punches from one animal
0 -300
Wavelength
400
500
(nm)
Fig. I. Spectral irradiance of the unfiltered measured with a radiospectrometer.
SE lamps as
were processed simultaneously but individually at 4°C. Care was taken not to include any tumors in samples. Subcutaneous fat was dissected away, and the skin was weighed before being minced into small pieces suspended in 8 ml of 0.05 M Tris (pH 7.5) containing 20% (w/v) NaCl, and immediately homogenized with a Polytron homogenizer at 4°C. The suspension was then centrifuged at 8000 g for 1 h. No significant amounts of soluble collagen were obtained during these extraction procedures. The resultant pellet was resuspended in 4 ml of a buffer containing 0.05 M Tris (pH 7.5) and 1.0 M NaCl, and was stirred for 3 days at 4°C. The samples was centrifuged at 8000 g for 1 h and the resultant supernatant dialyzed against water. These NaCl extraction procedures were repeated 3 times and the resultant 3 fractions were combined and then referred to as the neutral salt-soluble collagen (NSC) fraction. The pellet was washed with 4 ml of chilled 0.5 M acetic acid to remove NaCl, and immediately collected by centrifugation at 8000 g for 1 h. The pellet was resuspended in 4 ml of fresh 0.5 M acetic acid, and extracted for 3 days at 4°C. The suspension was then centrifuged at 8000 g for 1 h. The acetic acid extraction procedures were repeated 3 times and the resultant fractions were combined. Each combined supernatant and pellet was referred to as the acid-soluble collagen (ASC) fraction and insoluble collagen (ISC) fraction, respectively. Contents of collagen in each fraction were compared per fresh weight of tissues and per unit skin area. 2.5. Drtrminution of’ collagen content A sample of each fraction was lyophilized, and hydrolyzed in 6 M HCl at 110°C for 24 h. Hydroxyproline was determined by the method of Kivirikko et al. [17]. All quantitative assays were performed in triplicate. 2.6. Histology For light microscopy, skin specimens obtained from the dorsal skin were fixed with formalin and embedded in paraffin. Sections of the specimens (4-10 mm) were stained with hematoxylin and eosin (H&E), or by Van Gieson or Luna’s methods.
2.5 1
4 b&)
1.5-
.-P 5
OS-
o
T 4 week UVB start
IO
20
30
Age (weeks)
Fig. 2. Changes of wrinkle grade in control (0) and UVB-irradiated mice (0).
2.7. Stutistics Data were analyzed using Student’s t-test. Differences between regression lines were checked for significance by comparison of two regression slopes. The age and UVB influence were studied using analysis of linear correlation coefficients and statistical significance of the whole data. 3. Results Chronic UVB exposure caused no cornea1 opacities and no difference in body weight between the irradiated and age-matched non-irradiated control animals, while some tumors were observed on the exposed dorsal skin after 24 weeks. Therefore, for assessment of wrinkling, care was taken to exclude tumorous regions from skin samples. The first sign of fine wrinkling appeared in the dorsal skin of mice 4-5 weeks after UVB exposure. Some coarse wrinkles were observed after 6-8 weeks, and these were very apparent. 10 weeks after UVB irradiation (Fig. 2). Further UVB exposure increased the severity of wrinkling; wrinkles gradually became deeper and permanent and did not disappear upon movement of the animal while no wrinkling was observed in non-
80-
E .P 1 - ‘OaIN ;E u). 60‘= 0) E
g
-
50-
40
30:
1 0
t' 4 week UVB start
I 30
20
10
Age (weeks)
Fig. 3. Changes in tissue weight. When expressed as wet tissue weight per unit area of skin, significant ditTerences were obmice (0). served between control ( ) and UVB-irradiated
UVB-exposed age-matched control animals. These results observed in our HRjICR strain mice are in agreement with those of other reports in the Skh-1 strain [l 1,14,15]. Histological assessment using H&E and Luna staining showed hyperkeratosis, hypergranulosis, irregular epidermal hyperTable I Etfects of UVB irradiation Groups
Control UVB Control UVB Control UVB Control UVB Correlation coefficient
Period of irradiation
plasia, increases in elastic fibers and inflammatory infiltration in mice after irradiation at 24-weeksold. Parakeratosis was not always observed in the irradiated group. These findings indicate that photoaging of skin can be obtained in the HR/ICR mice as in the Skh-1 strains. Fig. 3 shows the effects of suberythemal dose UVB exposure on the fresh tissue weight per unit area of dorsal skin. UVB irradiation increased the tissue weight. The collagen of neutral salt-soluble (NSC) and acid-soluble (ASC) fractions were extracted from the UVB-irradiated animal skin, and the insoluble collagens (ISC) were the collected from the resultant residues. Tables 1 and 2 show the effects of UVB irradiation on hairless mouse skin collagen fraction per unit mass and per unit area. Significant decreases in ASC content were observed in the UVB-irradiated mice (per unit mass: 6-8 weeks; P -: 0.01, 11; P < 0.01, 16; P < 0.05, 24; P < 0.01, per unit area: 6-8 weeks; P < 0.01, 11; P < 0.05, 24; P < 0.01). In contrast, there were no differences between the irradiated and non-irradiated groups in NSC, ISC or total collagen content, except for a significant increase in ISC content per unit mass and total collagen content per unit area after 6-8 weeks of UVB exposure and significant decreases in ISC per unit
on hairless mouse skin collagen fractions II ,’
Collagen contents (/lg HYP:g wet tissue) i S.D.
weeks
3-6 (Av. 5.2) 6-S (Av. 6.2) II II I6 I6 20-23 (Av. 22.3) 24 Control
6 5 2 3 2 2 4 3 14
UVB
I3
ASC
NSC
ISC
Total
902 * I31 334* Ill** 468 i 39.4 165*61.4** 421 * 74.9 I03 * 19.7* 315 * 30.7 I22 * 59.0** I’ = - 0.949 P < 0.001 I’= -0.741 P < 0.005
88.5 i 66.4 72.2 & 60.9 73.3 * 14. I 71.8 i- 25.2 10.5 * 5.50 19.4 * 7.07 50.8 * 47.4 8.14 i 6.51 I’ = - 0.363
4491 k 659 5794 & 403** 5316+596 4519 k 197 7397* II9 4753 * 1074 5808 k 85.4 4088 i 227* I’ = 0.620 P < 0.01 I’ = ~ 0.778 P < 0.001
5481 + 798 6201 & 574 5858 _+650 4756 & 283 7829 i 199 4876 k I IO1 6174 i 874 4218i292’ I’= 0.417
I’= -0.613 P < 0.02
**,*Significantly ditrerent from age-matched control (P < 0.01, P < 0.05) .‘Indicates the number of mice included in each group.
I’= - 0.791 P < 0.001
Table 2 Effects of UVB irradiation Groups
Control UVB Control UVB Control UVB Control UVB Correlation coetticient
on hairless mouse skin collagen fractions
Period of irradiation (weeks)
II”
3-6 (Av. 5.2) 6-8 (Av. 6.2) II II I6 I6 20-23 (Av. 22.3) 24 Control
6 5 2 3 2 2 4 3 I4
UVB
I3
Collagen contents (j/g HYP cm’) f S.D. ASC
NSC
ISC
Total
39.3 * 6.5 18.1 2 5.4** 24.6 _+3.9 9.7 * 4.4* 16.8 + 3.3 749 * 2.0 17.0 k 2.6 9.7 * 4.!3** I’= - 0.893 P
3.8 &- 2.9 3.8 * 3.1 3.8 & I.0 4.2 f I.8 0.4 f 0.2 I .4 f 0.6 2.7 f 2.5 0.6 f 0.5 r = - 0.308
195.9 * 33.3 106.6 + 3s.2** 279.2 f 52.3 259.9 f 34.8 295.4 f 10.6 345.7 f 107.6 313.3 f 61.3 322.0 f 22.5 I’ = 0.788 P < 0.001 I’ = 0.204
239.1 *4O.l 337.9 f 2o.t3** 307.7 f 57.3 273.8 k4l.l 312.7 + 14.2 354.5 * 110.2 333. I i 64.2 332.4 i 27.7 r = 0.685 P i 0.005 I’= 0.089
r= -0.531 P < 0.05
**.*Significantly ditrerent from age-matched control (P < 0.01, P < 0.05). .‘lndi&tes the number of mice included in each group.
mass after 24 weeks and per unit area after 6-8 weeks and in total collagen content per unit mass after 24 weeks of UVB irradiation compared with age-matched controls. Especially, the ASC content both per unit mass (Fig. 4) and unit area (Fig. 5) decreasedexponen-
tially with age in the irradiated and control mice. The ASC content in control animals was always higher than that in the irradiated animals. The content of ASC was already reduced to approximately 30% of that in age-matched control animals by the first 6 weeks of irradiation. The 501
1200
4 week UVB start
Age (weeks)
Fig. 4. The acid-soluble collagen (ASC) content in both control (0) and irradiated mice (0) per fresh weight decreased with age: correlation coefficients were I’ = - 0.949 (P < 0.001) and I’ = - 0.741 (P < 0.005). respectively.
4 week UVB atart
Age (weeks)
Fig. 5. The acid-soluble collagen (ASC) trol (2) and irradiated mice (0) per creased with age; correlation coefficients i 0.001) and I’ = - 0.556 (P < 0.05).
content in both conunit area of skin dewere I’ = - 0.893 (P respectively.
decrease in ASC content in these irradiated mice occurred at the same time as wrinkle formation. 4. Discussion We found that wet tissue weight per unit area increases with age in both UVB-irradiated and control mice. The wet tissue weight of the irradiated animals was always higher than that of the controls. This may be responsible for the previously reported increases in skin thickness and skin water content [9] after UVB irradiation. Chatterjee et al. [9] reported that comparisons of biochemical data normalized to unit area and to unit mass of skin provide important information on the nature of the changes in skin components. We also used these comparisons in our collagen fraction analyses. In our study, IX and total collagen contents following UVB irradiation decreased when expressed per wet tissue weight but not per unit area, with the exception of ASC content. Chatterjee et al. [9] reported that total skin collagen content both of chronologically aged and UVBexposed mouse skin remained unchanged per unit area; however. significant decreases were observed per unit mass (wet weight) after 24 weeks of UVB irradiation. Our results support these findings. We observed that ASC contents, both per unit area and per wet tissue weight were significantly decreased after UVB irradiation compared with those of age-matched non-irradiated controls. A number of investigators have reported collagen content changes after UVB irradiation of mouse skin. In general. collagen contents decrease after UVB irradiation [ 18-221. We also observed an age-related exponential reduction in the content and the proportion of ASC from mouse skin. Similar age-related changes in ASC have been reported in human skin [23-251. Uitto [26] pointed out, through an in vitro study of human skin, that the reduced ASC content during aging concurs with the reduced rate of collagen synthesis. Recently, Schwartz et al. reported that the content of pN Type III collagen was reduced in sun-damaged skin [I] and UV-irradiated mouse skin [27], and they suggested that the decrease in pN type III
procollagen was due to increased degradation rather than reduced synthesis. The reduction of ASC contents after UVB irradiation was similar to those with aging; however, these changes may be generated by different mechanisms. The inflammatory infiltrate present in chronically sun-damaged skin is known to contain macrophages and mast cells [28]. These cells serve as reservoirs for proteinases such as collagenase and elastase that can modify the extracellular matrix of sun-damaged skin. Indeed, sparse infiltration was observed in the UV-irradiated mice in this study. The solubility of collagen from acute or subacute inflamed skin was lower than that from normal skin. whereas the turnover rate of collagen from inflamed tissues generally shows a high value [29]. Furthermore, the collagen of sun-damaged human skin has lower levels of the cross-linked nonreducible hydroxylysinonorleucine [no]. This abnormal cross-linkage pattern may decrease the solubility of collagen. The relative spectral contributions of FS 20SE lamps used in this study were 63. 36 and 0.5% in the UVB (280-320 nm). UVA (320-400 nm) and uvc ( <: 280 nm) regions. respectively. Therefore, we cannot exclude the possibility that UVA and:or IJVC also interact, either positively or negatively, with UVB. Recently, collagenase mRNA was shown to be induced in fibroblasts after irradiation [3 1.321. These results suggested that direct stimulation of collagenase synthesis in human skin fibroblasts by UVA irradiation many contribute to the connective tissue damage such as wrinkle formation. Compared to the changes in collagen types with UVB irradiation [S], the decrease in ASC shown in our study was a more marked phenomenon. The decrease in ASC content in the irradiated group was observed within the initial 6-week period. Furthermore. some coarse wrinkling was seen after approximately 6 weeks of UVB irradiation. These results suggested that the early quantitative changes of collagen with UVB exposure and the formation of wrinkles occurred concomitantly. Considering this relationship. we are inclined to assume that the decreased ASC content in UVB-irradiated skin may be caused by an increased rate of specific ASC degradation and/or
62
Y. T&emu et ul. ! Journal of‘ Dermatological
by decreasedsolubility of collagen with cross-linking, and that these qualitative changes lead to the formation of wrinkles on the hairless mouse back skin. A number of investigators have reported significant relationships between the formation of wrinkles and histological changes after UVB irradiation of hairless mouse skin [11,14]. Recently, Moloney et al. [15] reported that sufficient UVB irradiation will eventually cause changes in dermal elastin and collagen content; however, wrinkle formation precedes such changes. Our results indicated changes in structural proteins of the dermis occurring nearly simultaneously with wrinkle formation, and we consider these changes to have some effect on wrinkle formation, We believe that the study of wrinkling in animals will lead to clarification of the mechanism of wrinkle formation in humans. Further studies are required to clarify whether the changes in collagen, which are accelerated by UVB irradiation, are specific to hairless mice (HR/ICR), how these changes occur, and how they are related to wrinkle formation. References [I ] Schwartz E, Cruickshank FA, Christensen CC, Perlish JS Lebwohl M: Collagen alterations in chronically sun-damaged human skin. Photochem Photobiol 58: 841-844, 1993. [2] Chen VL, Fleischmajer R, Schwartz E, Palaia M, Timple R: Immunochemistry of elastotic material in sun-damaged skin. J Invest Dermatol 87: 3344337, 1986. [3] Oikarinen A. Karvonen J, Uitto J, Hannuksela M: Connective tissue alterations in skin exposed to natural and the serapeutic UV radiation. Photodermatology 2: 15-26, 1985. [4] Smith JG, Davidson EA, Sams WM, Clark RD: Alterations in human dermal connective tissue with age and chronic sun damage. J Invest Dermatol 39: 347-350, 1962. [5] Sams WM, Smith JG, Burk PC: The experimental production of elastosis with ultraviolet light. J Invest Dermatol 43: 467-471, 1964. [6] Nakamura KW, Johnston C: Ultraviolet induced connective tissue changes in rat skin: a histopathologic and histochemical study. J Invest Dermtol 51: 253-258, 1968. [7] Kligman LH, Akin FJ, Kligman AM: The contributions of UVA and UVB to connective tissue damage in hairless mice. J Invest Dermatol 84: 272-276, 1985. [8] Kligman LH, Gebre M, Alper R, Kefalides NA: Collagen metabolism in ultraviolet irradiated hairless mouse skin
Science I2 (1996) 56-63
and its correlation to histologic observations. J Invest Dermatol 93: 210-214, 1989. [9] Chatteriee RM. Benzinger J. Ritter JL. Bissett DL: Chronic ultraviolet B -radiation-induced biochemical changes in the skin of hairless mice. Photochem Photobiol 51: 91-97, 1990. UOI Johnston KJ, Oikarinen Al, Lowe NJ, Clark JG, Uitto J: Ultraviolet radiation-induced connective tissue changes in the skin of hairless mice. J Invest Dermatol 82: 5877590, 1984. Ull Bissett DL, Hannon DP, Orr TW: An animal model of solar-aged skin: histological, physical and visible changes in UV-irradiated hairless mouse skin. Photochem Photobiol 46: 367-378, 1987. 1121Bissett DL, Hannon DP, Orr TW: Wavelength dependence of histological, physical and visible changes in chronically UV-irradiated hairless mouse skin. Photochem Photobiol 50: 763-769, 1989. [I31 Bryce GF, Bogdam NJ, Brown CC: Retinoic acids promote the repair of the dermal damage and the effacement of wrinkles in the UVB-irradiated hairless mouse. J Invest Dermatol 91: 1755180, 1988. [I41 Kiss I, Chen S, Tramposch KM: The effect of high and low ultraviolet-B dose exposure on the degree of hairless mouse skin wrinkling. Photochem Photobiol 53: 109- 112, 1991. 1151 Moloney SJ, Edmonds SH, Giddens LD, Learn DB: The hairless mouse model of photoaging: evaluation of the relationship between dermal elastin, collagen, skin thickness and wrinkles. Photochem Photobiol 56: 505-511, 1992. ['61 Kim HJ, Bogdan NJ, D’Agostaro LJ, Gold LI, Bryce GF: Effect of topical retinoic acids on the levels of collagen mRNA during the repair of UVB-induced derma1damage in the hairless mouse and the possible role of TGF-b as a mediator. J Invest Dermatol 98: 3599363, 1992. ['71 Kivirikko KL, Laitinen LO, Prockop DJ: Modifications of a specific assay for hydroxyproline in urine. Anal B&hem 19: 249-255, 1967. [181Alpermann H, Vogel HG: Effect of repeated ultraviolet irradiation on skin of hairless mice. Arch Derm Res 262: 15-25 1978. [I91 Shuster S, Bottoms E: Effect of ultraviolet radiation on skin collagen of intact living mice. Nature 214: 599-600 1967. 1201Boyer B, Fourtanier A, Kern P, Labat-Robert J: UVAand UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice. J Photochem Photobiol B: Biol 14: 2477259, 1992. [21] Maeda K, Naganuma M, Fukuda M: Effects of chronic exposure to ultraviolet-A including 2% ultraviolet-B on free radical reduction systemsin hairless mice. Photochem Photobiol 54: 7377740, 1991. [22] Trautinger F, Mazzucco K, Konobler RM, Trenz A Kokoschka EM: UVA- and UVB-induced changes in hairless mouse skin collagen. Arch Dermatol Res 286: 490-494, 1994.
Y. Takema rl ul. i Journal of’ Dermatological [23] Bakerman S. Quantitative extraction of acid-soluble human skin collagen with age. Nature 196: 3755376, 1962. [24] Schnider SL, Kohn RR: Effects of age and diabetes mellitus on the solubility and nonenzymatic glucosylation of human skin collagen. J Clin Invest 67: 1630-1635, 1981. [25] Miyahara T, Murai A, Tanaka T, Shiozdwa S. Kameyama M: Age-related ditferences in human skin collagen: solubility in solvent, susceptibility to pepsin digestion and the spectrum of the solubilized polymeric collagen molecules. J Gerontol 37: 651-655, 1982. [26] Uitto J: Connective tissue biochemistry of the aging dermis. Dermatologic Clinics 4: 4333446, 1986. [27] Schwartz E, Cruickshank FA, Mezick JA, Kligman LH: Topical all-trans retinoic acid stimulates collagen synthesis in vivo. J Invest Dermatol 96: 9755978, 1991.
Scirnw
12 (1996) 56-63
PI Lavker RM, Kligman AM: Chronic heliodermatitis:
~291
[301
[311
[321
63
a morhphologic evaluation of chronic actinic dermal damage with emphasis on the role of mast cells. J Invest Dermatol 90: 3255330, 1988. Bailey AJ, Bazin S, Delaunay A: Changes in the nature of the collagen during development and resorption of granulation tissue. Biochim Biophys Acta 328: 383-390. 1973. Yamauchi M, Prisayanh P, Haque Z, Woodley DT: Collagen crosslinking in sun-exposed and unexposed sites of aged human skin. J Invest Dermatol 97: 938941, 1991. Petersen MJ, Hansen C, Craig S: Ultraviolet A irrddiation stimulates collagenase production in cultured human fibroblasts. J Invest Dermatol 99: 440-444, 1992. Schartfetter K, Wlaschek M, Hogg A, Bolsen K Schothorst A. Goerz G, Krieg T, Plewig G: UVA irrddiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch Dermatol Res 283: 506-51 I, 1991.
Dermatological Science
ELSEVIER
Journal of Dermatological
Science I2 (1996) 56663
The relationship between quantitative changes in collagen and formation of wrinkles on hairless mouse skin after chronic UV irradiation Yoshinori Takema, Michihiro Hattori*, Katsunori Aizawa’ Biological Sciencr Laboratories.
Tochigi, Kao Corporation
Received 26 April
2606 Akabane, Idikai,
Haga, Tochigi, 321-34 Japan
1995; revision received 2 June 1995; accepted 9 June 1995
Abstract Female albino hairless mice were irradiated chronically with sub-erythemal doses of UVB radiation. Collagen extracted from the irradiated or non-irradiated dorsal skin of mice was fractionated into neutral salt-soluble (NSC), acid-soluble (ASC) and insoluble fractions (ISC). An age-related exponential decrease in the content and proportion of acid-soluble collagen was found in each group. The contents and the proportions of ASC from irradiated mice were always significantly lower than those from age-matched control animals. Age-related slight decreases were observed in the contents (per fresh weight of tissues) of NSC, ISC and total collagen in the control group but decreases in these collagen contents after UVB irradiation were marked. A dramatic decrease in ASC occurred nearly concomitantly with wrinkle formation in the irradiated mice. The decrease of acid-soluble skin collagen in irradiated mice may play a role in the formation of wrinkles on hairless mouse dorsal skin. Kqwwdss: Acid-soluble
collagen; Wrinkle formation;
UVB;
1. Introduction Chronic UV exposure of human skin leads to alterations in the dermal connective tissue such as changes in relative composition and in deposition Ahbrr~icrtions: UVB: ultraviolet radiation in the wavelength range 290-320 nm, ASC: 0.5 M acetic acid-soluble collagen, ISC: insoluble collagen, NSC: neutral salt-soluble collagen, SDS: sodium dodecyl sulfate. * Corresponding author, Tel.: 285 68 7585: Fax: 285 68
7452. ’ Present address: National Nishisatonaka. Ohoteramachi,
Institute for Basic Biology, 38 Okasaki-City, Aichi. Japan
Hairless
pattern of the dermal matrix proteins collagen and elastin [l-4]. A number of investigators have reported successin the development of histological animal models for solar-aged skin [5-61. Of these animal models, hairless mice have been used extensively in studies of the effects of chronic UV irradiation [7-91. Some biochemical analyses of UV-exposed mouse skin have also been reported [5,8-lo]. Wrinkles develop on these mice in response to irradiation and are considered as indicators of chronic UV exposure or photo-aging [ 11- 151.Despite several reports concerning collagen metabolism in wrinkle effacement by retinoic
0923-1811~‘96~$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved SSDI 0923- I8 I 1(95)00467-7
mice
acid using this mouse model [16] there have been few studies of the relationship between the progressive changes in collagen and the formation of UVB-induced wrinkles in the skin in this model except on the total collagen and pepsin-soluble collagen contents [9,15]. Chatterjee et al. [9] reported that there was a relationship between the UVB-induced increase in the thickness of hairless mouse skin and the formation of wrinkles. On the other hand, Moloney et al. [IS] investigated the quantitative and qualitative changes in dermal collagen and elastin which occur in response to chronic ultraviolet irradiation and reported that sufficient UVB irradiation will eventually cause changes in dermal elastin and collagen content; wrinkle formation precedes such however, changes. Therefore, the present study was undertaken to investigate in more detail whether UVB irradiation changes the contents of skin collagen and is correlated with the formation of wrinkles on hairless mice. 2. Materials
and methods
2.1. Animds Female hairless HR/ICR mice were used in this study. This strain was established from a cross between a hairless strain, HR/HR (originally from Nisseiken Corporation, Japan), and a normal haired strain, HaM/ICR, and has been maintained in our laboratory by hairless brothernorma1 haired sister mating for several years. The animals were fed ad libitum and housed under conventional conditions at a controlled temperature (23 f 2°C) humidity (55 + 10%) and light (12 h light/l2 h darkness, without any ultraviolet emission). Animals were cared for in accordance with our institutional guidelines. 2.2. Rudiation sowce The hairless mice were separated into 2 groups (n = 15) at 4-weeks-old. One group was irradiated with UVB and the other served as a non-irradiated control group. Toshiba FL20SE lamps were used as a UVB source without any filtering. The distance from the lamps to the animals’ back was 35 cm. The irradiation energy was 0.42 mW/cm’ which was monitored with a UV-radiometer-305/
365D (Topcon Co. Ltd., Tokyo) at 305 nm (290320 nm) and MSR7000 radiospectrometer (Optical Science Co. Ltd.). Fig. 1 shows the spectral output of the UVB source. The mice were exposed 3 times each experimental week at a dose of 61 mJ&m’ (just below the erythemal dose) for 24 weeks yielding a total dose of 4.39 J/cm”. Two to five mice were housed per cage, and during exposure the animals could move around freely in the cages. At intervals of approximately 4 weeks after initiation of treatment, 2 or 3 animals in each group were killed and dorsal skin was excised for analysis. 2.3. Wrinkle nwusurenwnt Dorsal skin wrinkling caused by chronological suberythemal dose UVB exposure was graded each week according the method described by Bissett et al. [l l] as follows: grade 0, no coarse wrinkles; grade 1, a few shallow coarse wrinkles: grade 2, some coarse wrinkles; grade 3, several deep coarse wrinkles. The scale range from 0 for the normal animals to 3 for heavily wrinkled skin. 2.4. Step~ise extraction of collagen Animals were sacrificed by an overdose of chloroform, and the dorsal skin (1.57 cm’) was immediately collected. Five punches from one animal
0 -300
Wavelength
400
500
(nm)
Fig. I. Spectral irradiance of the unfiltered measured with a radiospectrometer.
SE lamps as
were processed simultaneously but individually at 4°C. Care was taken not to include any tumors in samples. Subcutaneous fat was dissected away, and the skin was weighed before being minced into small pieces suspended in 8 ml of 0.05 M Tris (pH 7.5) containing 20% (w/v) NaCl, and immediately homogenized with a Polytron homogenizer at 4°C. The suspension was then centrifuged at 8000 g for 1 h. No significant amounts of soluble collagen were obtained during these extraction procedures. The resultant pellet was resuspended in 4 ml of a buffer containing 0.05 M Tris (pH 7.5) and 1.0 M NaCl, and was stirred for 3 days at 4°C. The samples was centrifuged at 8000 g for 1 h and the resultant supernatant dialyzed against water. These NaCl extraction procedures were repeated 3 times and the resultant 3 fractions were combined and then referred to as the neutral salt-soluble collagen (NSC) fraction. The pellet was washed with 4 ml of chilled 0.5 M acetic acid to remove NaCl, and immediately collected by centrifugation at 8000 g for 1 h. The pellet was resuspended in 4 ml of fresh 0.5 M acetic acid, and extracted for 3 days at 4°C. The suspension was then centrifuged at 8000 g for 1 h. The acetic acid extraction procedures were repeated 3 times and the resultant fractions were combined. Each combined supernatant and pellet was referred to as the acid-soluble collagen (ASC) fraction and insoluble collagen (ISC) fraction, respectively. Contents of collagen in each fraction were compared per fresh weight of tissues and per unit skin area. 2.5. Drtrminution of’ collagen content A sample of each fraction was lyophilized, and hydrolyzed in 6 M HCl at 110°C for 24 h. Hydroxyproline was determined by the method of Kivirikko et al. [17]. All quantitative assays were performed in triplicate. 2.6. Histology For light microscopy, skin specimens obtained from the dorsal skin were fixed with formalin and embedded in paraffin. Sections of the specimens (4-10 mm) were stained with hematoxylin and eosin (H&E), or by Van Gieson or Luna’s methods.
2.5 1
4 b&)
1.5-
.-P 5
OS-
o
T 4 week UVB start
IO
20
30
Age (weeks)
Fig. 2. Changes of wrinkle grade in control (0) and UVB-irradiated mice (0).
2.7. Stutistics Data were analyzed using Student’s t-test. Differences between regression lines were checked for significance by comparison of two regression slopes. The age and UVB influence were studied using analysis of linear correlation coefficients and statistical significance of the whole data. 3. Results Chronic UVB exposure caused no cornea1 opacities and no difference in body weight between the irradiated and age-matched non-irradiated control animals, while some tumors were observed on the exposed dorsal skin after 24 weeks. Therefore, for assessment of wrinkling, care was taken to exclude tumorous regions from skin samples. The first sign of fine wrinkling appeared in the dorsal skin of mice 4-5 weeks after UVB exposure. Some coarse wrinkles were observed after 6-8 weeks, and these were very apparent. 10 weeks after UVB irradiation (Fig. 2). Further UVB exposure increased the severity of wrinkling; wrinkles gradually became deeper and permanent and did not disappear upon movement of the animal while no wrinkling was observed in non-
80-
E .P 1 - ‘OaIN ;E u). 60‘= 0) E
g
-
50-
40
30:
1 0
t' 4 week UVB start
I 30
20
10
Age (weeks)
Fig. 3. Changes in tissue weight. When expressed as wet tissue weight per unit area of skin, significant ditTerences were obmice (0). served between control ( ) and UVB-irradiated
UVB-exposed age-matched control animals. These results observed in our HRjICR strain mice are in agreement with those of other reports in the Skh-1 strain [l 1,14,15]. Histological assessment using H&E and Luna staining showed hyperkeratosis, hypergranulosis, irregular epidermal hyperTable I Etfects of UVB irradiation Groups
Control UVB Control UVB Control UVB Control UVB Correlation coefficient
Period of irradiation
plasia, increases in elastic fibers and inflammatory infiltration in mice after irradiation at 24-weeksold. Parakeratosis was not always observed in the irradiated group. These findings indicate that photoaging of skin can be obtained in the HR/ICR mice as in the Skh-1 strains. Fig. 3 shows the effects of suberythemal dose UVB exposure on the fresh tissue weight per unit area of dorsal skin. UVB irradiation increased the tissue weight. The collagen of neutral salt-soluble (NSC) and acid-soluble (ASC) fractions were extracted from the UVB-irradiated animal skin, and the insoluble collagens (ISC) were the collected from the resultant residues. Tables 1 and 2 show the effects of UVB irradiation on hairless mouse skin collagen fraction per unit mass and per unit area. Significant decreases in ASC content were observed in the UVB-irradiated mice (per unit mass: 6-8 weeks; P -: 0.01, 11; P < 0.01, 16; P < 0.05, 24; P < 0.01, per unit area: 6-8 weeks; P < 0.01, 11; P < 0.05, 24; P < 0.01). In contrast, there were no differences between the irradiated and non-irradiated groups in NSC, ISC or total collagen content, except for a significant increase in ISC content per unit mass and total collagen content per unit area after 6-8 weeks of UVB exposure and significant decreases in ISC per unit
on hairless mouse skin collagen fractions II ,’
Collagen contents (/lg HYP:g wet tissue) i S.D.
weeks
3-6 (Av. 5.2) 6-S (Av. 6.2) II II I6 I6 20-23 (Av. 22.3) 24 Control
6 5 2 3 2 2 4 3 14
UVB
I3
ASC
NSC
ISC
Total
902 * I31 334* Ill** 468 i 39.4 165*61.4** 421 * 74.9 I03 * 19.7* 315 * 30.7 I22 * 59.0** I’ = - 0.949 P < 0.001 I’= -0.741 P < 0.005
88.5 i 66.4 72.2 & 60.9 73.3 * 14. I 71.8 i- 25.2 10.5 * 5.50 19.4 * 7.07 50.8 * 47.4 8.14 i 6.51 I’ = - 0.363
4491 k 659 5794 & 403** 5316+596 4519 k 197 7397* II9 4753 * 1074 5808 k 85.4 4088 i 227* I’ = 0.620 P < 0.01 I’ = ~ 0.778 P < 0.001
5481 + 798 6201 & 574 5858 _+650 4756 & 283 7829 i 199 4876 k I IO1 6174 i 874 4218i292’ I’= 0.417
I’= -0.613 P < 0.02
**,*Significantly ditrerent from age-matched control (P < 0.01, P < 0.05) .‘Indicates the number of mice included in each group.
I’= - 0.791 P < 0.001
Table 2 Effects of UVB irradiation Groups
Control UVB Control UVB Control UVB Control UVB Correlation coetticient
on hairless mouse skin collagen fractions
Period of irradiation (weeks)
II”
3-6 (Av. 5.2) 6-8 (Av. 6.2) II II I6 I6 20-23 (Av. 22.3) 24 Control
6 5 2 3 2 2 4 3 I4
UVB
I3
Collagen contents (j/g HYP cm’) f S.D. ASC
NSC
ISC
Total
39.3 * 6.5 18.1 2 5.4** 24.6 _+3.9 9.7 * 4.4* 16.8 + 3.3 749 * 2.0 17.0 k 2.6 9.7 * 4.!3** I’= - 0.893 P
3.8 &- 2.9 3.8 * 3.1 3.8 & I.0 4.2 f I.8 0.4 f 0.2 I .4 f 0.6 2.7 f 2.5 0.6 f 0.5 r = - 0.308
195.9 * 33.3 106.6 + 3s.2** 279.2 f 52.3 259.9 f 34.8 295.4 f 10.6 345.7 f 107.6 313.3 f 61.3 322.0 f 22.5 I’ = 0.788 P < 0.001 I’ = 0.204
239.1 *4O.l 337.9 f 2o.t3** 307.7 f 57.3 273.8 k4l.l 312.7 + 14.2 354.5 * 110.2 333. I i 64.2 332.4 i 27.7 r = 0.685 P i 0.005 I’= 0.089
r= -0.531 P < 0.05
**.*Significantly ditrerent from age-matched control (P < 0.01, P < 0.05). .‘lndi&tes the number of mice included in each group.
mass after 24 weeks and per unit area after 6-8 weeks and in total collagen content per unit mass after 24 weeks of UVB irradiation compared with age-matched controls. Especially, the ASC content both per unit mass (Fig. 4) and unit area (Fig. 5) decreasedexponen-
tially with age in the irradiated and control mice. The ASC content in control animals was always higher than that in the irradiated animals. The content of ASC was already reduced to approximately 30% of that in age-matched control animals by the first 6 weeks of irradiation. The 501
1200
4 week UVB start
Age (weeks)
Fig. 4. The acid-soluble collagen (ASC) content in both control (0) and irradiated mice (0) per fresh weight decreased with age: correlation coefficients were I’ = - 0.949 (P < 0.001) and I’ = - 0.741 (P < 0.005). respectively.
4 week UVB atart
Age (weeks)
Fig. 5. The acid-soluble collagen (ASC) trol (2) and irradiated mice (0) per creased with age; correlation coefficients i 0.001) and I’ = - 0.556 (P < 0.05).
content in both conunit area of skin dewere I’ = - 0.893 (P respectively.
decrease in ASC content in these irradiated mice occurred at the same time as wrinkle formation. 4. Discussion We found that wet tissue weight per unit area increases with age in both UVB-irradiated and control mice. The wet tissue weight of the irradiated animals was always higher than that of the controls. This may be responsible for the previously reported increases in skin thickness and skin water content [9] after UVB irradiation. Chatterjee et al. [9] reported that comparisons of biochemical data normalized to unit area and to unit mass of skin provide important information on the nature of the changes in skin components. We also used these comparisons in our collagen fraction analyses. In our study, IX and total collagen contents following UVB irradiation decreased when expressed per wet tissue weight but not per unit area, with the exception of ASC content. Chatterjee et al. [9] reported that total skin collagen content both of chronologically aged and UVBexposed mouse skin remained unchanged per unit area; however. significant decreases were observed per unit mass (wet weight) after 24 weeks of UVB irradiation. Our results support these findings. We observed that ASC contents, both per unit area and per wet tissue weight were significantly decreased after UVB irradiation compared with those of age-matched non-irradiated controls. A number of investigators have reported collagen content changes after UVB irradiation of mouse skin. In general. collagen contents decrease after UVB irradiation [ 18-221. We also observed an age-related exponential reduction in the content and the proportion of ASC from mouse skin. Similar age-related changes in ASC have been reported in human skin [23-251. Uitto [26] pointed out, through an in vitro study of human skin, that the reduced ASC content during aging concurs with the reduced rate of collagen synthesis. Recently, Schwartz et al. reported that the content of pN Type III collagen was reduced in sun-damaged skin [I] and UV-irradiated mouse skin [27], and they suggested that the decrease in pN type III
procollagen was due to increased degradation rather than reduced synthesis. The reduction of ASC contents after UVB irradiation was similar to those with aging; however, these changes may be generated by different mechanisms. The inflammatory infiltrate present in chronically sun-damaged skin is known to contain macrophages and mast cells [28]. These cells serve as reservoirs for proteinases such as collagenase and elastase that can modify the extracellular matrix of sun-damaged skin. Indeed, sparse infiltration was observed in the UV-irradiated mice in this study. The solubility of collagen from acute or subacute inflamed skin was lower than that from normal skin. whereas the turnover rate of collagen from inflamed tissues generally shows a high value [29]. Furthermore, the collagen of sun-damaged human skin has lower levels of the cross-linked nonreducible hydroxylysinonorleucine [no]. This abnormal cross-linkage pattern may decrease the solubility of collagen. The relative spectral contributions of FS 20SE lamps used in this study were 63. 36 and 0.5% in the UVB (280-320 nm). UVA (320-400 nm) and uvc ( <: 280 nm) regions. respectively. Therefore, we cannot exclude the possibility that UVA and:or IJVC also interact, either positively or negatively, with UVB. Recently, collagenase mRNA was shown to be induced in fibroblasts after irradiation [3 1.321. These results suggested that direct stimulation of collagenase synthesis in human skin fibroblasts by UVA irradiation many contribute to the connective tissue damage such as wrinkle formation. Compared to the changes in collagen types with UVB irradiation [S], the decrease in ASC shown in our study was a more marked phenomenon. The decrease in ASC content in the irradiated group was observed within the initial 6-week period. Furthermore. some coarse wrinkling was seen after approximately 6 weeks of UVB irradiation. These results suggested that the early quantitative changes of collagen with UVB exposure and the formation of wrinkles occurred concomitantly. Considering this relationship. we are inclined to assume that the decreased ASC content in UVB-irradiated skin may be caused by an increased rate of specific ASC degradation and/or
62
Y. T&emu et ul. ! Journal of‘ Dermatological
by decreasedsolubility of collagen with cross-linking, and that these qualitative changes lead to the formation of wrinkles on the hairless mouse back skin. A number of investigators have reported significant relationships between the formation of wrinkles and histological changes after UVB irradiation of hairless mouse skin [11,14]. Recently, Moloney et al. [15] reported that sufficient UVB irradiation will eventually cause changes in dermal elastin and collagen content; however, wrinkle formation precedes such changes. Our results indicated changes in structural proteins of the dermis occurring nearly simultaneously with wrinkle formation, and we consider these changes to have some effect on wrinkle formation, We believe that the study of wrinkling in animals will lead to clarification of the mechanism of wrinkle formation in humans. Further studies are required to clarify whether the changes in collagen, which are accelerated by UVB irradiation, are specific to hairless mice (HR/ICR), how these changes occur, and how they are related to wrinkle formation. References [I ] Schwartz E, Cruickshank FA, Christensen CC, Perlish JS Lebwohl M: Collagen alterations in chronically sun-damaged human skin. Photochem Photobiol 58: 841-844, 1993. [2] Chen VL, Fleischmajer R, Schwartz E, Palaia M, Timple R: Immunochemistry of elastotic material in sun-damaged skin. J Invest Dermatol 87: 3344337, 1986. [3] Oikarinen A. Karvonen J, Uitto J, Hannuksela M: Connective tissue alterations in skin exposed to natural and the serapeutic UV radiation. Photodermatology 2: 15-26, 1985. [4] Smith JG, Davidson EA, Sams WM, Clark RD: Alterations in human dermal connective tissue with age and chronic sun damage. J Invest Dermatol 39: 347-350, 1962. [5] Sams WM, Smith JG, Burk PC: The experimental production of elastosis with ultraviolet light. J Invest Dermatol 43: 467-471, 1964. [6] Nakamura KW, Johnston C: Ultraviolet induced connective tissue changes in rat skin: a histopathologic and histochemical study. J Invest Dermtol 51: 253-258, 1968. [7] Kligman LH, Akin FJ, Kligman AM: The contributions of UVA and UVB to connective tissue damage in hairless mice. J Invest Dermatol 84: 272-276, 1985. [8] Kligman LH, Gebre M, Alper R, Kefalides NA: Collagen metabolism in ultraviolet irradiated hairless mouse skin
Science I2 (1996) 56-63
and its correlation to histologic observations. J Invest Dermatol 93: 210-214, 1989. [9] Chatteriee RM. Benzinger J. Ritter JL. Bissett DL: Chronic ultraviolet B -radiation-induced biochemical changes in the skin of hairless mice. Photochem Photobiol 51: 91-97, 1990. UOI Johnston KJ, Oikarinen Al, Lowe NJ, Clark JG, Uitto J: Ultraviolet radiation-induced connective tissue changes in the skin of hairless mice. J Invest Dermatol 82: 5877590, 1984. Ull Bissett DL, Hannon DP, Orr TW: An animal model of solar-aged skin: histological, physical and visible changes in UV-irradiated hairless mouse skin. Photochem Photobiol 46: 367-378, 1987. 1121Bissett DL, Hannon DP, Orr TW: Wavelength dependence of histological, physical and visible changes in chronically UV-irradiated hairless mouse skin. Photochem Photobiol 50: 763-769, 1989. [I31 Bryce GF, Bogdam NJ, Brown CC: Retinoic acids promote the repair of the dermal damage and the effacement of wrinkles in the UVB-irradiated hairless mouse. J Invest Dermatol 91: 1755180, 1988. [I41 Kiss I, Chen S, Tramposch KM: The effect of high and low ultraviolet-B dose exposure on the degree of hairless mouse skin wrinkling. Photochem Photobiol 53: 109- 112, 1991. 1151 Moloney SJ, Edmonds SH, Giddens LD, Learn DB: The hairless mouse model of photoaging: evaluation of the relationship between dermal elastin, collagen, skin thickness and wrinkles. Photochem Photobiol 56: 505-511, 1992. ['61 Kim HJ, Bogdan NJ, D’Agostaro LJ, Gold LI, Bryce GF: Effect of topical retinoic acids on the levels of collagen mRNA during the repair of UVB-induced derma1damage in the hairless mouse and the possible role of TGF-b as a mediator. J Invest Dermatol 98: 3599363, 1992. ['71 Kivirikko KL, Laitinen LO, Prockop DJ: Modifications of a specific assay for hydroxyproline in urine. Anal B&hem 19: 249-255, 1967. [181Alpermann H, Vogel HG: Effect of repeated ultraviolet irradiation on skin of hairless mice. Arch Derm Res 262: 15-25 1978. [I91 Shuster S, Bottoms E: Effect of ultraviolet radiation on skin collagen of intact living mice. Nature 214: 599-600 1967. 1201Boyer B, Fourtanier A, Kern P, Labat-Robert J: UVAand UVB-induced changes in collagen and fibronectin biosynthesis in the skin of hairless mice. J Photochem Photobiol B: Biol 14: 2477259, 1992. [21] Maeda K, Naganuma M, Fukuda M: Effects of chronic exposure to ultraviolet-A including 2% ultraviolet-B on free radical reduction systemsin hairless mice. Photochem Photobiol 54: 7377740, 1991. [22] Trautinger F, Mazzucco K, Konobler RM, Trenz A Kokoschka EM: UVA- and UVB-induced changes in hairless mouse skin collagen. Arch Dermatol Res 286: 490-494, 1994.
Y. Takema rl ul. i Journal of’ Dermatological [23] Bakerman S. Quantitative extraction of acid-soluble human skin collagen with age. Nature 196: 3755376, 1962. [24] Schnider SL, Kohn RR: Effects of age and diabetes mellitus on the solubility and nonenzymatic glucosylation of human skin collagen. J Clin Invest 67: 1630-1635, 1981. [25] Miyahara T, Murai A, Tanaka T, Shiozdwa S. Kameyama M: Age-related ditferences in human skin collagen: solubility in solvent, susceptibility to pepsin digestion and the spectrum of the solubilized polymeric collagen molecules. J Gerontol 37: 651-655, 1982. [26] Uitto J: Connective tissue biochemistry of the aging dermis. Dermatologic Clinics 4: 4333446, 1986. [27] Schwartz E, Cruickshank FA, Mezick JA, Kligman LH: Topical all-trans retinoic acid stimulates collagen synthesis in vivo. J Invest Dermatol 96: 9755978, 1991.
Scirnw
12 (1996) 56-63
PI Lavker RM, Kligman AM: Chronic heliodermatitis:
~291
[301
[311
[321
63
a morhphologic evaluation of chronic actinic dermal damage with emphasis on the role of mast cells. J Invest Dermatol 90: 3255330, 1988. Bailey AJ, Bazin S, Delaunay A: Changes in the nature of the collagen during development and resorption of granulation tissue. Biochim Biophys Acta 328: 383-390. 1973. Yamauchi M, Prisayanh P, Haque Z, Woodley DT: Collagen crosslinking in sun-exposed and unexposed sites of aged human skin. J Invest Dermatol 97: 938941, 1991. Petersen MJ, Hansen C, Craig S: Ultraviolet A irrddiation stimulates collagenase production in cultured human fibroblasts. J Invest Dermatol 99: 440-444, 1992. Schartfetter K, Wlaschek M, Hogg A, Bolsen K Schothorst A. Goerz G, Krieg T, Plewig G: UVA irrddiation induces collagenase in human dermal fibroblasts in vitro and in vivo. Arch Dermatol Res 283: 506-51 I, 1991.