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Topical Tretinoin Increases the Tropoelastin and

Fibronectin Content of Photoaged Hairless


Mouse Skin
Elaine Schwartz and Lorraine H. Kligman*
Departments of Dermatology, Mt. Sinai School of Medicin e, N e w Y o rk, N ew Y o rk; and ' University of Pennsylvania School of
Medicine, Philadelphia, Pelwsylvania, U .S.A.

Topical tretinoin treatment of photoaged hairless


mice has been shown in previous studies to stimulate
formation of a subepidermal zone of new connective
tissue characterized by enhanced collagen synthesis.
The aims of this study were to localize and/or quantify elastin, fibronectin, and glycosaminoglycans in
the same model. Hairless mice (Skh-1) were irradiated thrice weekly for 10 weeks with gradually increasing doses of ultraviolet (up to 4.5 minimal erythema doses per exposure) from Westinghouse FS-40
bulbs. Mice were then treated five times a week with
either 0.05% tretinoin, the ethanol:propylene glycol
vehicle, or nothing for another 10 weeks. Controls
included mice sacrificed after 10 weeks of ultraviolet
treatment and age-matched untreated animals. The

distribution of elastin and fibronectin was examined


by immunofluorescence microscopy, which revealed
fine fibrils in the subepidermal zone in tretinointreated skin. A quantitative slot-blot immunobinding
assay showed that tretinoin induced a threefold
higher amount of tropoelastin compared with controls. Insoluble elastin content (desmosine levels) was
similar in all groups. Although fibronectin content
was increased by ultraviolet radiation, tretinoin
treatment induced the largest increase. In contrast,
the amount of glycosaminoglycans, although increased by UVB radiation, was reduced by tretinoin
treatment. Key words: UV radiatioulglycosami,wglyca.usl
elastinldesmosiue. ] Invest Dermatol 104:518-522, 1995

uring the development of the h airless mouse mod el


for hum an photoaging, it was dis covered that
within a 10 - 1 5-week period after ultraviolet radiation (UVR) , the damaged dermal m atrix underwent partial and spon tan eo us restora tion [1,2].
Histologic and ultrastru ctural studies demonstrated that topical
treatmen t with tretinoin (all-trans-retinoic acid) in th e post-UVR
period accelerated tlus process by enhancing the production of a
subepidermal repair zon e of collagen [3,4] . A similar repair zone
was reported by Bryce et at [5] using either tretinoin or isotre tinoill
in a different strain (I-IRS/J) of photoaged mice.
More recently, using immunofluorescence mi croscopy and immunochemical and biochemical tecluliques, we reported that tretinoin enhanced col\age n synthesis in the photoaged hairless mouse.
The newly synthesized collagen was locali zed in the histochemically defined repair zone [6]. Using a UVR schedul e similar to that
of Kligman et at [3], other workers reported increased collagen
synthesis after 6 weeks of treatment with tretinoin [7]. In addition,
a two- to fo urfold increase in stea dy-state mRNA leve ls for types I
and III collagen was induced in the photoaged mouse by tretinoin
[6,8] and isotretinoin treatments [8].
UVR-induced changes in the non col\agenous derm al matrix

have been documented ex te nsive ly. Increased amounts of elastic


fib ers, fibronectin, and glycosaminoglycans hav e been reported in
th e photoaged hairless mouse and in human skin by histochemical
[1,9-13] , immullochemi cal [14,15] , and biochemical mean s
[14,16,17] . However, there is little information co ncerning the
e ffe ct of tretinoin on th e non coLlage nous d ermal matrix in photoaged skin. One study of the hairless mouse reported that tretinoin
treatment did not affect glycosaminoglycan synthesis [18]. A seco nd
study fail ed to find an e ffe ct of tretinoin on the UVR.-:induced
desmosine content [5] . R ecent studi es on hum an photoa ged skin
hav e focused mainly on the clinical e ffe cts of tretinoin [19, 20] or on
collagen deposition [21 ,22] . The only reports of treti noin-indu ced
changes in noncoLlagenous prote ins i.nvol ved the epidermis or cell
culture res ults. Lunden et (// [23] reported that 6 months of tretinoin
treatment did not alter hyaluroni c acid content in th e epidermis or
in blister fluid . Increased amounts of fibron ectin were reported b y
V ar31u el al [24 - 26] in fibroblast cultures, whereas in keratino cyte
cultures, fibron ectin synthesis was d ecreased after tretinoin treatment.
The aim of t1us study was to d etermine the effects of topical
tretinoin on the elastin , fibronectill, and g lycos3 minoglyc;1ll contents of the photoaged hairless m o use d e rmi s.

Manuscript received August 10, 1994; revised becember 17, 1994;


accepted for publication December 27, 1994.
Tills paper was presented ill part at the ESD R -JS ID-SID T ri contin c nwl
Meeting in May 1990.
Reprint requests to : Dr. Lorra.ine H . Kligman, Dcpartmcnt of Derma to 1ogy, Univers ity of Pennsylvania School of Med icin e, 415 C urie Boulevard,
Suite 227, Philadelp hia, PA 19104-6142 .

MATERIALS AND M ETH ODS


Animals and Irradiation Schedules O ne hundred Skh-hairless-1 (albin o) female mi ce, ages 6-8 weeks (C harles River Laboratories, Wilmingto n, MA) were ho used individu all y with free access to food and water.
R oo m Lightin g (12-h on-off cycle) was with Gencral Electric F40 GO go ld
Allorcscent bulbs, which emit n o UVR. Fo ur gl OUpS of20 mi ce each wcre

0022-202X/95/S09.50 SSD I0022-202X(95)00001-2 Co pyright

518

'1995 b y T he Society fo r In vestigative Dermatology, In c.

VOL. 104 , N O . 4

A PRIL 1995

exposed to UVB radiation , whereas the fifth gro up served as age-matched,


unirradiated controls.
The VVR so urce was a bank of eight W es tingho use FS- 40 "su nl amps"
(280-400 nm ; peak irradian ce at 313 run) . Lamps were positio ned 16 cm
above the mi ce for the thrice-weekl y e xposures, w hi ch continued fo r 10
weeks . Each UV R. dose, reached gradua lly o ver the first 4 w eeks b y
in crements of 0.5 minimal erythema doses (MED), wa s 4 .5 MED (0 .07
] / cm 2 VVB). Irradiance was measured with an IL 700 R.esearch R.adiome te r
(International Ligh t, Inc ., N ewburyport, MA) using a UVB sensor with
peak sensitivity at 290 nm.
p o st_Irradiation Treatments At the end of the 10-w eek irradiation
period, one irradiated group was sacrificed to provide tissue for the
VVR-only baseline valu es. The remaining three groups were treated as
fo llows: 1) no furth er treatment, 2) 0.05% treti noin in an ethanol:propylene
glycol vehicle (70:30 v/v) containing 0.05% butylated hydro,,:ytoluene, and
3) v ehicl e. Dorsa l applica tions (100 ILl each) were m ade five times a week
for 10 weeks .
B iopsi es and Histologic Stains The mice were sacrificed by cervical
dislocation, and dorsal trunk skin was excised and frozen at - 70C until
used. An adjacent strip of skin was prepare d for light m icroscopy and stained
with Luna's al dehyde fuchsin [27] to monitor depth of the repair zon e.
Mowry' s coll oidal iron stain was used to assess glycosaminoglycan con tent.
Immunoflu orescence M i croscopy Frozen sections (8 /km) w ere
treated with cold acetone for 5 min and rinsed with phospbate-buffered
saline. The specimens were then incubated for 30 min at 37C with ither
rabbit anti-bovine elastin or rabbit anti-mouse fibrone ctin antibodies,
washed with phosphate-buffered saline, and treated for 30 min at 37C with
goat anti-rabbit IgG conjugated with fluores cein isothio cyanate (Organon
T e knika-Cappel, W es t C hester, PA) . Controls used IgG prepare d from the
sera of nonimm u nized rabbits .
Tropoel astin and Insoluble El astin Anal yses Equa l w e t w e igh ts of
skin were homogenized and defatted with but3nol:diisopropyl ether (40:60
v/v) . Previous experimen ts have determined that tropoelastin is not extra cted by this procedure . Extraction was overnigh t at room temperatu re,
with stirring, in 0.05 M Tris HCI (PH 7.5), 1 % sodium dodecylsulfate, and
0.33 M mercaptoethanol containing proteolytic in hi bitors. T his w as fo llowed by centrifugation at 10,000 rpm for 30 min . The supernatants were
electrophoretica lly concentrated fivefo ld at 2.5 mA /sample for 90 min
(Little Blue Tank, Isco, Lincoln, NE) and analyzed for tropoelastin b y a
quantitative inllnunobinding assay [28] . Dilutions of extracts and standards
were immobi lized o n a nitroce llu lose sheet. T he sheet wa s th en blocked
with bovine serum albu min , washed, an d incubated with antibodies directed
against bov ine alp ha e lastin. T he sheets w e re again washed and treated with
1251_labeled protein A. After a tl~ird washing, the nitrocell ulose wa s dried
and exposed to fi lm w ith intensifying screens. T he fi lms were scanned usin g
a Beckman DU8 Spectrophotometer (550 nm) .
T he insoluble residues were washed extensively , lyophilized , and hydrolyzed in 6 N HCl for 72 h at 107C to re lease the d esmosines. H ydrolysates
were 'Ipp lied to J1lini-cell ul ose co lumns an d til e desmosines were eluted ,
dried, and analyzed by high perforJ1lance liquid chromatography [29] .
Fib ronectin Analysis Fibronecti n was quantifi ed as described previou sly
[30]. Five skins from e ach group were individually trimmed to equal surface
area, homoge l~i zed , d efatted , and extracted for 48 h w ith 5 m1l100 m g dry
weight of 0.0 5 M Tris HCI (PH 7.5) , 1.0% sodium d o decylsulfate, 0.33 M
mercaptoethanol, and prote olytic inhibitors (1 /kg/ml aprotinill, 2 mM
p henylmethylsulfony l fluorid e, '\ mM N-e thyl ma le imide). The latte r are
serine protease inhibitors, and sodium dodecylsu lfate is an effective general
protease inhibitor. Many of the pla sma enzymes are serine proteascs,
incl uding elastase. T he suspension was centrifuged at 10,000 rpm for 30
min . Supernatan ts were electrophoretically concen trated fivefo ld at 2.5
mA/sampl e for 90 min (lsco Inc. ) .
Fibronectin content was ana lyzed by a specific en zyme-linked immunosorben t assay using rabbit anti-mouse fibron ectin and purified mous e
fibron cctin (Chcmicon , Temecula, C A), as fo llows. Mou se fibronectin (400
ng) was added to w ells in a flat-bottom mi crotite r p late and allowed to
incubate overnight at room temperatu re. To w e lls in a row1d-bottom
micro titer plate , 11 0 ILl of rabb it anti-mou se fibro nectin antigcn (diluted
1:15,000) and 110 /kl of dil utions ofeitller standard mou se fibronectin (1.25
/kg/ Ill 1 diluted to 1 ng/ ml) or samples were mixed for 1 h at room
temperature, incubated for 1 h at 37C , and then kept overni ght at 0-4 C.
Excess antigen was removed from thc fla t wells, w hich w e re then washed ,
and 0.35 1111 of 1'Yo bovine serum al bull1in wa s add ed. After 1 h , the bovine
serum albumin was removed , the w e ll s were washed, and 20 0 /kl from the
rou nd well s was 'Idd ed . T he plate was in cubated at 37C for 45 min an d
washed, and anti- rabbit IgG-liJlked alka l.ine ph osphatase (1 :1000 diluti o n)

TRETIN O I N

M ODU LATES NON - C OLLAGENOUS DERMAL MATRlX

519

was adde d to the wells. After incubation at 37C for 1 h and washing, the
wells were rinsed rapidly with substratc buffe r (10% diethanolamine, pH
9.8), and 0 .2 ml of a soluti on of substrate p-nitrophenyl phosphate (1
m g/ ml) in substrate buffer was added. T he reaction was allowed to proceed
at 37C for 15-30 min and was terminated by the add itio n of 0.1 ml ofl N
Na O H . Products wcre quantified by reading of the plate at 405 nm.
Appropri ate controls lacking antige n , antibody, enzY'ne, or substrate were
ru n simultaneously.
Glycosaminoglycan Analysis Glycosa m inoglycans were q uan tified as
described previously [30]. Weighed skins were minced, defatted in acetone:
ether (1:1), dried, and suspended in 10 ml of 0. 1 M phosphate buffer (PH
6.5) with 0.005 M ethylenediamine tctraacetic acid and 0.005 M cysteine
HCI. Digestion was with papain (50 ml containing 24 IU / ml; Sigma
C hemica l Co., St. Lo uis, MO) for 24 h at 65 C. T he digest was diluted to
40 ml with water and ce ntrifuged for 2 h at 12,000 rpm (0-4 C). A 1%
suspension of cetylpyridinium chloride was added dropwise to the supernata nt [31]. After storage overnight at 4C, the suspension was centri fuged
at 12,000 rpm for 1 h at 0-4 C. T he precipitate was dissolved ill 300 /kl of
n-propano l and 75 ILl of 1% cetylpyridinium chloride. T he glycosaminoglyca ns were further precipitated with 5 ml of absolute etll a.n o l and a few drops
of sodium acetate. T he precipitatc was washed with etllan ol and diethyl
e th er, a.ir dried , redissolve d in 1 ml of water, transferred to preweighed
tubes, and dried, after which the glycosamin oglycan weight was calculated.
Glycosaminoglycans w ere redissolved in 1 ml of water, and the uronic acid
conte nt was determined by the carbazole assay [32].

RESULTS
Histochemical Findings Confirm Tretinoin Enhancement
of Dermal Matrix Deposition Lun a's stain revealed the subepidennal zone of new collagen in all specimens at 10 weeks after
UVR, as reported in previou s studi es [3 ,4] . In tretinoin-treated
skin, the zone was con side rably deeper, m o re extensive across the
specim ens , and be tter de lin eated , with co mpressed elastosis at the
lower border (data not shown) . Mowry's stain showed increased
g lycosal11inoglycans in all specimens. Histologically, the increases
induced b y UVR alo n e appeared to regress too slowly in th e
post-UVR period to m ake histologic quantification of the effect of
tretinoin diffi cult (data not shown) .
Imn:lullofluorescencc and IInnlunobinding Studies Locate
and Qua.ntify Tretinoin-Enhallced Tropoelastin Accumulation
1111111 II II ojillorescel/ce: Sections from normal, 10-weeks UV-in-adiated, post-UVR untrea ted , and vehicle-treated skins showed similar patchy f1uorescences throughout the dermis (Fig in). After 10

Figure 1. Detection of tropoelastin in the subepidermal dermis by


inlnlullofluorescencc with antibodies directed against bovin e c l astin. a) VVR for 10 weeks. Flu o rescen ce is evident in diffuse th ick and thin
fibers throughout tllC de rmis. AIT{)",/,eans. dermoepidermal junction . Bar, 10
/km . b) UVR. for 10 weeks followed by trctino in for 10 weeks. An intensel y
stained band of e lastosis delineates the lower border of the band of unsta ined
new coll agen (aIHI"'s). Within ti,e dark band are thi n fluores cent sO'ands of
ela stin . Aml/II/wans , d ennoepiderlllal junction. Bar, 10 ILm.

520

THE JOURNAL OF INVESTIGATI VE

SCHWARTZ AND KLIGMAN

weeks ofUVR treatment with tretinoin, fluorescent strands of new


elastic tissue were present in the mainly unstained subepidermal
dermis (Fig lb), which correspo nd s to the morphologically defined
repair zone [3,4] . A concentration of patchy fluorescence was
present at the lower border of the zone, corresponding to the
compressed elastosis seen histologically. Fluorescence was not
visible in controls treated with normal rabbit serum.
1ll1nllmobi"dillg Assny: The autorad iogram of the slot- blot immunobindi.ng assay using antibodies against bovine alpha elastin and
12sI-labeled protein A clearly showed an increase in tropoelastin in
tretinoin - treated skin (Fig 2). Densitometric scanning of the autoradiogram films showed that the increase was approximately threefold higher than in any of the other treatment groups (Table I).
High Fe,fonllallce Liquid Chrom afogmpil.y Jor Insoillble Elastin: Determination of desmosine content illdicated no sign ificant increase in
insoluble elastin in any of the groups (Table I). The values ranged
from 10.1 to 11.9 ng desmosine/mg wet weight.

Tretinoin-Enhanced Fibronectin Content is Demonstrated


by Immunofluorescence and Enzyme-Linked Immunosorbent Assay
1m """lOjhlOrescellce: Fluorescent staining was more abundant but
otherwise similar to that seen for elastin in normal, 10-week
UV-irradiated, post-UVR untreated, and vehicle-treated skins (Fig
3a). After 10 weeks of UVR treatment with 0.05% tretinoin, the
subepidermal re pair zone was filled with intensely stained flJ1e
fibrillary fibronectin (Fig 3b) . Fluo rescence was not visible in
controls treated with normal rabbit serum.
Qllallfificatio/1 by E llz yme-Lillked ],"""I,,osorbeu.t Assay: W h eth er
caJc ulated by area, wet weight, or dry weight, UVR significa ntl y
in creased fibronectin le vels compared with norm al controls (Table
II). T hese levels decreased during the lO-week post-UVR period
with vehicle or no topical treatment, but remained significantly
higher than in normal controls. The greatest increase was induced
by tretinoin (more than twofold).

Tretinoin Diminishes the UVR-Induced Increase in Glycosaminoglycans Quantification by the carbazole method showed

Table I.

DEI~ATOLOGY

Effect of UVR and Tretinoin on Elastin


Accumulation"

Treatment Group
I. Untreated
II. UVR 10 weeks

111111111Jlobinding Assay
(ng/elastin/mg wet
weight)"

Desmosine
(ng/mgwet
weight)"

150 16
175 39

11.4 1. 2
10.93.8

198 29

10.1 2.3

184 29

10.4 0.6

479 62'

11.9 1.1

Ill. UVR 10 weeks; untreated

10 weeks
UVR. 10 weeks; vehicle
10 weeks
V. UVR 10 weeks; tretinoin
10 weeks

TV.

Hairless mice were irradiated with UVll for 10 weeks and then treated with either
0.05% tretinoin . the vehicle. or nothing. Skins were homogenjzed . def:.'tted. extracted.
and analyzed for ttopoclastin by a quantitative irnmunobinding assay [28J. Insoluble
residues were hydrolyzed in 6 N I-I C I. and desmosines we re eluted o n milli-ceLlulose
columns. dried, and analyzed by high performance liquid chromatography [29).
II Mean :: SEM; n = 5.
f P = 0.0025 versus untreated contro ls, Student t tcst.
(I

that glycosaminoglycan levels were signifi cantly increased after 10


weeks of UVR. The levels nonnalized in the post-UVR p eriod
with vehicle or no topical treatment. However, tretinoin treatment
resulted in a significant lowering of glycosaminoglycan levels when
compared with normal or 10-week UVR co ntrol s (Table III).
Quantification of g lycosaminoglycans by total weight using
cetylp yridin ium chl oride precipitation revealed no significant differences between any of the treatment groups. On the basis of skin
area, values ranged from 482 to 628 /Lg/cm 2, with an average SEM
of 40 . Calculation s based on wet or dry weight showed an eve n
more narrow ran ge (data !Jot shown).
DISCUSSION
Previous hi sto logi c [3-5], immunochemical, and biochemical studies [6,7] have shown that topical tretinoin treatment of photoaged
h airless mouse skin stim ulates the deposition of a subepidermal
repair zone of newly synthesized colla gen. This study expands the
ill "i"o findings, demonstrating a tretilloin-induced threefold increase in soluble e lastin and a greater than twofold increase in
fibronectin. ]n agreement with Bryce ef al [5], we failed to find a

A
B

I I I

I I

I I I

Figure 2. Increased content oftropoelastin in extracts of tretino intreated, photodamaged skin by immunobinding assay. Autoradiogram shows seq uential dilutions of extracts. A, UVR and tretinoin-trcatcd;
B, UVR and vehicle-treated; C, UVR and untreated; D, VVR only; E,
untrea ted.

Figure 3. Detection of fibronectin ill the subepidermal dermis by


immunofluorescence with antibodies directed against mouse 6bronectill. a) UVR for 10 weeks. Fluorescence highlights a diffuse network
offibronectin throughout the dermis. AI1'owheads, demlOepidermaljunction.
Bar, 10 p.m. b) UVR for 10 weeks followed by tretinoin for 10 weeks. Dense
parallel fluorescent strands of fibronectin fill the subepiderma.l (5E) band of
new conn ective tissue. Note that fibronectin in the lower dermis retains the
network appea.rance. Arrowheads, dermoepidermaI junction. Bar, 10 /-Lm .

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Table II.

521

Effect of UVR and Tretinoin on Fibronectin Content"

Treatment Group

ng/cm 2

I. Untreated
II. UVR 1 0 weeks
III. UVR 10 wceks; untreated 10 weeks
IV. UVR 10 weeks; vehicl c 10 weeks
V . UVR 10 w ecks; tre tinoin 10 wceks
Com parisons/'
1 vers us lJ
r vers us III
) versus )V
I vers us V
Jl vers us III
\J vers us IV
11 versus V
JI) versus IV
111 versus V
)V versus V

64.4 2.3
119.5 19 .6
73.9 3 .3
88 .5 4.5
160.9 23.9
p
p
p
p
p

n g/ mg Wet Wcight
0.80
1 .39
1.20
1.39
2.08

<
<
<
<
<

0.025
0.05
0.0025
0.005
0 .05
NS
NS
p < 0.025
P < 0.005
P < 0.01

P
P
P
P

<
<
<
<

P
P
P
P

<
<
<
<

ng/mg Dry Weigh t


3.64 0.28
7.32 1.1
4. 99 0.21
6.72 0.52
10.38 1.1 6

0.06
0.22
0.03
0.08
0.24

0.025
0.0125
0.0005
0.0025
NS
NS
0.05
0.05
0.0025
0.025

P
P
P
P
P

<
<
<
<
<

P
P
P
P

<
<
<
<

0.01
0.005
0.0025
0.0005
0.05
NS
0.025
0.01
0.0125
0.0025

Hairless mice were irradia ted with UVll for 10 weeks ;m d th en treated with e ither 0.0 5% trcli noi n . th e vchicle . or n othing fo r to weeks. Skins \vere extracted and th e am ounts
of fibroll cctin were dctcnnincd by a speci fi c enzym e-linked il11ll1UIlOSorbcllt :lssay. as d escribed in AlJntcri(J (s ami /\fl etllOds. D:ltjl arc m ean :t SEM.
I, Statistical analysis: Student t test (11 = 5). NS. Il o t sig nifi ca n t .
(I

Table III.

Effect of UVR and Tretinoin on Glycosaillinoglycan Content"


G lycosaminoglycans (n = 5)

Treatm cnt Gro up


J. Untrcated
11. UVR 10 wee ks
111. UVR 10 weeks; untreated 10 weeks

IV . UVR 10 weeks; ve hicl e 10 weeks


V . UVIl 10 weeks; trctin o in 10 wce ks
.
/,
Companso ns
) versus 11
I vers us III
I ve rsus IV
I versus V
11 ve rsus III
II versus IV
II versus V
II) vers us IV
III vers us V
)V versus V

Ca rbazole Meth o d (p.g/cm


21.77 0.72
29.6 2.9
2 1.68 1.63
21.88 3.39
14.76 2.52
p

<

0.025
NS
NS

P < 0.025
< 0.025
NS
P < 0.005

NS

<

0.025

NS

p.g/ m g Wet Weight

0.286
0.329
0.314
0.303
0. 196

0.018
0.024

=
0.009
0.039
0.038

NS
NS
NS
P < 0.05
NS
NS

P < 0.0 1
NS
P < 0.0 1
P < 0.05

p.g/ m g Dry Wcigh t


1.54 0.10
1.87 0.13
1. 73 0.043
1.71 0.233
1.09 0.207
p

< 0.05

NS
NS
P < 0.05
NS
NS

P < 0.01
NS
P < 0.01
P < 0.05

,I Hairless mice we re irrad iated with UVI3 fo r 10 weeks ~lI1 d thell tre ated with either 0.05% trcti ll o in , the ve hicle. or n othin g: for to weeks. SkillS were min ced . d Cf.1ttcd. and
d igested with p :,p;ljn. G lycosaminoglycOl ns we re precipitated with 1% cetylpyridiniulli chlo ride. T he uro nic Olcid co nte lll' w as d etermined by th e carbazole assay 13 2] . Dam arc mean
:!: SEM .
1/ Statistical 'H1al ys is: Stude nt t tes t. NS. Hot sig nifi c:lIl t .

tretinoiJl-induced cban ge i.n insolubl e e la stin as m easure d b y desmosine conte n t in the lO-week post-UVR period. In con trast to
our findings , Bryce el nl [5] reported that insolubl e elastin was
increased as a result of the UVR exposures. T he most like ly reason
for this divergence lies in the di fferent protocols. T he 5- 6 months
of UVR used b y Bryce et nl to produ ce photoagillg in th e HRS /J
mice , co mp ared with our 10-week sch eduJ e, allowed more time for
tropoelastin to accumu late and become cwsslinke d into matUl'c
fibers.
The small bu t signifi ca n t increase in glycosa minog lycaJ1s after 10
weeks of UVR was expected on th e basis of histochemi cal ev aluation . However, the considerabl e d ec rease "vith tretino in treatmen t
was not anticipated . This fmding supports th e premise of C hen et nl
[18] that glycosaminoglyca ns may not be responsible for th e
wrinkle effacement reported in th ese mi ce with tretinoin [5] . T hese
au th ors found no increase in sul fate d glycosaminoglycal1 or byaluronic acid synth esis in explants of UV - irradiated, tretinoin-treate d
hairless mouse skin as compared with irradiated, vehi cle-treated
contwls. Unfortunately, the effect of 10 w e eks of irradiation alone
cou ld not be assessed beca use C hen et nl [1. 8] did not include these
or 110rmal controls in their study.
Other studies with hairl ess mi ce exanun ed eith er UVR alone or

treti noin alon e . In agreem e nt with our findings o n th e effect of


UVR alone, M a.rgelin et nl [33] found increased glycosaminoglyca.n
synth esis in explan ts ITom UV-inadiated hairl ess mi ce. However,
the increase was seen onl y in the proteoglyca ns h eparin and heparan
sul fute. Synthesis of dermatal1 su lfute and hya luronic ac id was not
affec ted . An ill lI i llO study [34] that exa mined lifetime application of
tretinoin to unirradiated h airless mi ce d emons trated histochemically a large increase in d erm al and epidermal glycosaminoglycans.
Taken together with o ur current findings, the eviden ce suggests
that UVR. may increase some classes of glycosa minoglyca.ns,
wh ereas tretinoi.tl acts to decrease UVR-induced levels. However,
in unil'radiated skin, tretinoin may increase tlus dermal matrix
co mpon en t [34].
T he difficulty of obtaining suffi cient human biopsy material h as
limited exte nsive bioch emi cal studies of tretinoin effects on nonco Uagenous d ermal m atrix. T he few ill IJil ro studi es h ave examined
the effec ts ofUVR or tretinoin, but not the combination of the two.
For exaUlple, Schwartz and C rui cksh ank,!, irradiated normal human

'1' Sc hwartz, C ruickshank: Elastin, fibronectin and coll agen synthesis in


UV irradja ted d ermal fibrob la sts (abstr). J Il1 /1csl Dcnlln lol 96:586, 1991.

522

dermal fibrobla sts il'l vitro in the absence of tretinoin and found
increased synthesis of both fibronectin and tropoelastin, a finding
co nfirmed in the present ill vivo study. As a second exa mpl e, an in
vivo study examined the effect of tretino in on sun-protected buttock
skin. Fisher e/. til [35] reported a tretil10in-induced increase in the
transform ing growth fuctor-J3-induced "mucin" or glycosa mil1oglycans and proteoglycans. Thus, there is sparse biochemical
information on human studi es with whi ch to co mpare the effect of
tretinoin on noncollagenous dermal matrix in the photoaged
hairless mouse .
It has been establish ed that man y of the UVR-induced changes in
the hairless mouse correlate well with those occurring in human
photoage d skin [36]. As trials of tretinoin-treated human skin
approach and exceed 2 years, it is becoming apparent that the
hairless mouse mod el is also a predictive one for the repair of
human photoaging. Recent reports have demonstrated a tretinoininduced subepidermal repair zone of new collagen [19,37], along
with reduction in the abundant glycosaminoglycans present in
photoaged skin [37]. The ultrastructural reappearan ce of type VII
coll agen anchoring fibrils has been reported after 2 years of
treatment [21]. Most recently, Griffiths e/ al [22] demonstrated
increased intracellular and extracellular type I coll agen synthesis in
the papillary de rmi s after 12 months of tretinoin treatment. In
acco rdance with its short life span, m etabolic processes occur more
rapidly in mi ce than in hum ans. It is there fore likely that with time,
mu ch of what h as been dis co vered in tretinoin-treated photoaged
mouse skin will be confirmed in human skin.

T ltis reseal. 1t was SIIppol1ed ill pat1 by a gralll frO Ill lite R ./4/. J " /IIISO II Pltarlllacel/lical R esearch IlIslill/le, Raritall, Nelli J ersey.
14/e Ilt allk MarilY IIJ. Crasb) Sally Adall' s, alld K erlllil 14/lttll1oll fo r II, e enre alld
'reallllel'lt of the allilllais; Dorotlty C all1pbell fo r lec/Illieni ltislocltelllisl'l' expe,1ise;
alld Brellda Lace). for typillg ,lte lIIallllscripl.

REFERENCES
I.
2.

3.

4.

5.

6.
7.

8.

9.

10.

11 .
12.

THE J OURNAL OF INVESTIGATIVE DERMATOCOGY

S HWARTZ AND K LI GMAN

Kli gman LH . Akin FJ . Kligman AM: Prevention of ul trav iolct damage to I'he
dcmlis of hairl ess mice by sunscreens. J IlI lIcst D el'l l/ fl'o' 78 : t 8 1- 189. 1982
Kl1 g m.m LH. Akin FJ. Kl ig m an AM : Sunscree ns pro m ote repa ir of ul t rilviolct
radiatio n- indu ced dermal damage.) ',, "csl Oer",nlOl 8 1:98-1 0 1, 1983
Klignl:ln LH. C hen HD . Kligman AM : Topic:1I rc tin o ic acid e nhan ces the rcp;lir
of ultraviolet damaged dermal connective tiss l1 e. CC",,, CCI Tissue Res 12: 139150, 1984
Klig man Ll-l: Effects of a ll-tran s rc tinoic acid o n the d ermis of hairJess mice. ) A m
Awn Oenlllllol 15:779-785. 1986
I3ryce GF, 130gdan NJ. I3rown CC: Retin oic acids pro m ote the repa ir of the
dermal d:unagc and the effacemenl of w rinkles in the UVB- irradia tcd hairl ess
mouse .) " /IIcsl Oe,.", nlol 9 1: 175-180. 1988
Schwartz E. C ru ickshank I' A. M ezick JA. Kligma n LH: Topica l all- trans rctinoi c
acid stimulates collage n synthesis ill I/ iIJO . J IlI lIc'S I DCrlllIltol 96:975-978, 199'1
C he n S. Ki ss I. Tramposch KM : Eff"ects of all-trans-retin oic acid on UVI3irradiated and no n- irradiated hairl ess m Ollse skin .) tllilest D en1laloI9 8:248-254 .
1992
Kim HJ , Bogdan NJ. D 'Agostaro LS. Gold L1 , Bryce GF: Effe ct of topical rctinoic
acid on the Icvels of collagcn mRN A d urin g repair of UVB-induced de rmal
damage in the h airl ess m Ou se and th e possibl e ro le of TGF-/3 as a mediator.
) 1""eSI Oem,nlol 98 :359-363. 1992
l3i ssett DL. H annon Dr>, O rr TV: An anim"lmo dcl o f solar aged skin. Histo logic.
physical and visible changes in chro nic:111 y UV- irr:1dia tcd hairl ess mouse skjn.
P/lOllIc/""" I'l,olobiol 46:367-378, 1987
Fourca nie r A. I3crrc bi C. C harles N. Mcda isko . N OQlc t JI': I"AlIc nce of UV
li ght o n natu ral ski n :1ging in hairl ess mi ce. Co.wH,tir Dt:rwato/l :289-300, 1986
Sanls WM. Smith J G: T h e histoche mistry of chro ni ca ll y sun damaged skin .
) "IIIesl Oe,.,,,olol 37:447-452. '1964
Kli gma n AM : Earl y destructive CfCCLS o f ~ unli g ht o n hum:111 skin . JAMA
2 10:2377-2380, 1969

'13.

M enter JM, Pearl WS. Yadven MW. Willis I: UV-indu ccd elevation of , kin
glycosaminoglyciln levels. I: Rapid meas urement of sk.in glycosarninoglycans
by titration w ith acridine o ran ge. P/"'loc/,c", I'l,olobiol 38:481-486, '1983
14 . Schwanz E: Connective tissue alterations in the skin of ul traviole t irradiated
hairl css mice.) "/IIcsl Ocmlnlol 9 1: 158-161 , 1988
15. C hen VL, Fleischmajer R., Schwartz E. Palaia M, Timpl R : Immunochemistry of
elastotic material in sun damaged ski_n .] flwcs( DemUl(ol 87:334 -337. 1986
16. Smith J G . Davidson EA. Taylor R.W : Human cutaneous Illucopolysaccharides:
the e ffects of age and chro nic sun damage . In : Montagna W (cd .). A dlJnllCCS ill
Biology of Skirr. Vol, 6, Aging. 1965, pp 2 11-2 '18
17. C hatterjee I~, B enZinge r MJ. Ritter JL, Bissett DL: C hro nic ultraviole t B
radiation- i.nduccd biochclnica l chan ges in thc skin of hairless 1nice. PllOtocilcm
P/rolobiol 51 :91-97. 1990
l 8 . C hen S, Beehler B, Sugimoto G, Trarnposch KM: Ellects of aU-trans retino ic acid
o n glycosaminoglyca n synthesis in photodamaged h airless m o use skin . J I,ll/cst
O emonlol 101 :237-239 , 1993
19. Kligman AM. Grove GL, Hirose I~. Leyden JJ: Topical tretinoin for photoaged
skin.) A ", Acml Oe,."onlol 15 :836-83 9. 1986
20 . Ellis C N, Weiss JS , Hamil ton TA. H eadington J T, Zelickson AS , Voorhees JJ:
Su stained improve m ent with prolonged topical tretiJloin (retinoic acid) for
photoaged skin .) Alii Acnd O el1lonl,,1 23:629-637, 1990
21 . Woodley DT, Zelickson AJ.Briggaman RA, H amilton TA, WeissJA, Ellis C N.
Voo rhees JJ: T reatment of photoaged skin with topical trctinoin increases
deTlnal-epidermal anch or ing f,brils. A preliminary report. )AMA 263:30573059. 1990
22. Griffith s CEM. Russman AN. Majmudar G , Singer R S, Hamilton TA, Voorhees
JJ : Restoration of collagcn form:ltion in photod:lmaged human skin by
trctinoin (retinoic acid). N E llgl) Mcd 329:530-535, 1993
23. Lunden A. B erne B, Mi chaelsson G: To pica l retinoic acid treatment of photo aged
skin: its efi'ccts on h yalurol1 an distributio n in epidermis and on hyaluro n:ln and
rctino ic acid in suction b lister Auid. ACln D erlll Vell ereol (Slocklt) 72:423-427,
1992
24 . Varani J . Nickoloff" BJ, Dixit VM. Mi tra RJ, Voorhees )J : AII-tra"s retinoie acid
stimulates growth of ad ult human keratillocytes culturcd in growth facto rdeflciellt mcdium , inhibits produ ction of thrombospondin and fibron ectin. :lnd
redu ces adhcsion . ) "/llesl Oer",nlol 93:449-454, 1989
25. Varani J. Mitra R S, Gibbs D. Plum SH. Dixit VM. Mitra RJr, Wang T, Siebert
KJ . Nickoloff BJ . Voorhees JJ : All trans retin oic acid stimulates growth and
extraccllular matri x prod uction in growth-inhibited cultured human skin
f,brobla sts.) [lilies, Oemon,,,1 94:717-723, 1990
26. Varoni J . Astrom A. Griffiths CEM . Voorhees JJ: Induction of proliferation of
growth inhibited ke ratinocytes : lIld fibrobla sts in mo no laycr culturc by sodium
lau ry l sulfate: comparison with all-tnms rctino ic acid.] IUl/cs t O e,."w(oI 97:91792 1. 1991
27. Kli gman LH: Luna' s techniqu c-" bea utiful st:lin [or clastin . A m) De,.",atoptl tllOl
3:199-20 1. 198 1
28 . J ahn R. Schiebler W, Grce ng:lrd P: A quantit:ltive dot-inununo binding assay for
pro te ins usin g nitrocellulosc me mbrane fi lters. Proc N atl Awd Sci USA 81:
1684 - 1687 . 1984
29. Schwartz E, C rui ckshank FA , Lebwohl M : Determination of desl1losincs in
clasti_n - rclatcd skin disorders by isocratic high pcrform:lll ce liquid chromatography. Ex/, Mol Poll, ol 52:63-68. 1990
30. Sch wartz E. M ezickJA . Gelldil'ne lli co GJ. Kligman LH: The {II viiI{) prevc11 tio n of
corti costeroid-indu ced skin atrophy by trcti_u oin in th e hairless m ouse is
accornpanicd by m od ulatio n o f collagc lI. glycosa min oglyc:lI1s . and fibrOl1cctin .
) IlI vcsl 0 ",."",10/10 2:241-246. 1994
3 '1. ltoss SC. Osbo rn TG. Dorner ltW' Z ucker J : Glycosa rninoglycan content in the
skin of the tight-skin mouse. A rtlrrilis Rite"", 26:653-657 . 1983
32 . Bitter T, Muir HM : A m odified uronic acid carba zole reaction. A ,wl BiochclII
4:330-334.1962
33. Margc1iu 0 , Fourtaniel' A, T hcvClllll T. M edaisko C , Breton M, Picard J :
Alteration s of pro tco glycans in ultra vio let irradi :lted skin. PlwwchclII Pir%lJiol
58:211 -218, 1993
34. Kligma n LH , M ezick J A, Capeto la RJ. T horn e EG: Lifetirne topi cal application
of trctin oin to h ai rl ess rnice . Arlo Oem, Vell ereol (Slockl,) 72:4 18-422, 1992
35. Fisher GJ , Tavakko l A, Griffi ths CEM, Elder J T, Z hang Q-Y. Finkel L,
DanielpDur 0, Glick AB. Highley H. Ellings w orth l, Voorhees JJ : Differential
m odula tion ofttan sfo nning gro wth f.1ctor- 131 expressio n and mucin dcp ositio n
by retino ic ;Icid and sodium ~a lJryJ sul f:'ltc jn hUJnan skin . J IImesJ Derma/oJ
98: 102-108. '1992
36 . .K1igman LH : Thc hairless m Ollse ~lI1d ph o tO:lging : yearl y review . PIIlI/Oc/'CW
Pltol,r/JioI54:1109 - 111 8 ,1991
37 . KJi g man AM , Gra ham GF: H isto l ogi c~11 cha_l1gcs in fac ial skin aftc r dajly
application of trcti no in for 5 to 6 years.) O erlll Trenllll e,,1 4: 11 3-117 . 1993