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Regular Articles |
From the Departments of Pathology*
and
Dermatology,
The University of Michigan
Medical School, Ann Arbor, Michigan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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65
and 57%, respectively) in photodamaged forearm skin compared
to sun-protected hip skin. We next investigated whether reduced type I
procollagen production was because of inherently reduced capacity of
skin fibroblasts in severely photodamaged forearm skin to synthesize
procollagen, or whether contextual influences within
photodamaged skin act to down-regulate type I procollagen synthesis.
For these studies, fibroblasts from photodamaged skin and
matched sun-protected skin were established in culture. Equivalent
numbers of fibroblasts were isolated from the two skin sites.
Fibroblasts from the two sites had similar growth capacities and
produced virtually identical amounts of type I procollagen protein.
These findings indicate that the lack of type I procollagen synthesis
in sun-damaged skin is not because of irreversible damage to fibroblast
collagen-synthetic capacity. It follows, therefore,
that factors within the severely photodamaged skin may act in some
manner to inhibit procollagen production by cells that are inherently
capable of doing so. Interactions between fibroblasts and the
collagenous extracellular matrix regulate type I procollagen synthesis.
In sun-protected skin, collagen fibrils exist as a highly
organized matrix. Fibroblasts are found within the matrix, in
close apposition with collagen fibers. In photodamaged skin,
collagen fibrils are shortened, thinned, and
disorganized. The level of partially degraded collagen is 3.6-fold
greater in photodamaged skin than in sun-protected skin, and
some fibroblasts are surrounded by debris. To model this
situation, skin fibroblasts were cultured in
vitro on intact collagen or on collagen that had been partially
degraded by exposure to collagenolytic enzymes. Collagen that had been
partially degraded by exposure to collagenolytic enzymes from either
bacteria or human skin underwent contraction in the presence of dermal
fibroblasts, whereas intact collagen did not. Fibroblasts
cultured on collagen that had been exposed to either source of
collagenolytic enzyme demonstrated reduced proliferative capacity (22
and 17% reduction on collagen degraded by bacterial collagenase or
human skin collagenase, respectively) and synthesized less type
I procollagen (36 and 88% reduction, respectively, on
a per cell basis). Taken together, these findings indicate that
1) fibroblasts from photoaged and sun-protected skin are similar in
their capacities for growth and type I procollagen production; and 2)
the accumulation of partially degraded collagen observed in
photodamaged skin may inhibit, by an as yet unidentified
mechanism, type I procollagen synthesis.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reduced procollagen levels in severely sun-damaged skin may result from irreversible, UV-induced damage to the cellular and molecular machinery governing collagen synthesis and breakdown in skin fibroblasts. Alternatively, damaged extracellular matrix may act in some manner to down-regulate procollagen synthesis by resident skin fibroblasts. It is known in this regard that interactions between collagen peptides and cell surface integrins induce intracellular signaling events,11-14 and that procollagen synthesis in fibroblasts is regulated by these interactions.15-18 In the presence of degraded collagen fibers, intracellular signaling is disrupted.19,20 To distinguish between these two possibilities, we have assessed type I procollagen synthesis by human fibroblasts in photodamaged skin in vivo, and by fibroblasts from photodamaged skin cultured in vitro. In addition, we have determined the level of partially degraded collagen in photodamaged human skin relative to sun-protected skin, and have examined the effect of partially degraded collagen on type I procollagen synthesis. The data reported herein demonstrate that collagen fragmentation is increased, and that type I procollagen synthesis is reduced in sun-damaged human skin compared to sun-protected skin. Despite this, fibroblasts isolated from severely photodamaged skin possess similar capacity for in vitro procollagen synthesis as fibroblasts from sun-protected skin. Furthermore, in vitro type I procollagen synthesis is reduced in the presence of partially degraded collagen relative to intact collagen. Taken together, these data suggest that reduced collagen synthesis in photoaged skin occurs as a result of contextual influences, including the effects of damaged collagen on dermal fibroblast function.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A total of 42 individuals (22 males and 20 females) participated in this study. All of the individuals were characterized by the presence of severe photodamage on their forearms based on clinical criteria (eg, coarseness of the skin and degree of wrinkling). The age range was 46 to 83 years with the average age being 69 years. Replicate 4-mm full-thickness punch biopsies of forearm and sun-protected hip skin were obtained from each individual. All procedures involving human study subjects were approved by the University of Michigan Institutional Review Board, and all study participants provided written informed consent before their inclusion in the study. It should be noted that in 18 of the individuals who participated in this study, we were able to obtain biopsies of sun-protected underarm skin as well as skin from the other two sites. Overall, sun-protected skin from the underarm and sun-protected hip skin were very similar in regards to the parameters assessed (ie, collagen fragmentation, fibroblast isolation rates, proliferation, and collagen synthesis; see below).
Electron Microscopy
Skin biopsies from forearm and hip skin were fixed overnight in 4% electron-microscopic grade glutaraldehyde in 0.1 mol/L of cacodylate buffer (Sigma, St. Louis, MO) at pH 7.4. After fixation with 2% osmium tetroxide (EM Sciences, Fort Washington, PA) buffered in 0.1 mol/L of cacodylate buffer, sections were dehydrated with graded alcohol to 2x 100% alcohol and 2x propylene oxide (EM Sciences). The samples were embedded in pure epon resin. One-µm tissue sections were cut, stained with Toluidine blue, and examined at the light microscopic level. Ultrathin sections were cut from areas of interest, stained with lead citrate and uranyl acetate (all from EM Sciences), and observed in a Phillips 400 transmission electron microscope.
Assessment of Collagen Degradation in Human Skin
Skin biopsies from forearm and hip skin were homogenized in Tris
buffer (20 mmol/L, pH 7.3) and centrifuged. The pellet, containing the
collagenous extracellular matrix, was resuspended in 150 µl of Tris
buffer containing 75 µg of 
-chymotrypsin, and incubated for 8
hours at 37°C. The pellet from homogenized skin biopsies incubated in
buffer alone served as control. At the end of the incubation period,
the reaction tubes were centrifuged at 10,000 x g for
10 minutes. Supernatants were collected and assayed for hydroxyproline
using automated amino acid analysis. Unlike intact fibrillar collagen,
partially degraded collagen can be further broken down and the
hydrolysis products liberated from tissue by
-chymotrypsin.21
The amount of released collagen
hydrolysis product can be determined by measurement of hydroxyproline,
which is a modified amino acid present in collagen but rarely found in
other proteins.22
Assessment of Type I Procollagen Synthesis in Human Skin in Vivo
Assays for type I procollagen mRNA and protein were used to
identify and quantify collagen-elaborating cells in skin samples. Type
I procollagen (1) gene expression was assessed by in situ
hybridization. Fresh skin samples were immersed in OCT and frozen in
liquid nitrogen. Frozen sections (6 µm) were hybridized with
digoxigenin-labeled antisense and sense type I procollagen 1 cRNA
probes, as described previously.23
Cells expressing type I
procollagen (1) mRNA were quantified by counting under light
microscopy. Type I procollagen protein was assessed by immunohistology.
Frozen sections (6 µm) were stained with either one of two mouse
monoclonal antibodies (SP1.D8, and M38) to human type I procollagen
(1 chain) and an immunoperoxidase-conjugated secondary
antibody.7,10
The SP1.D8 antibody was developed by Dr.
Heinz Furthmayr and obtained from the Developmental Studies Hybridoma
Bank under the auspices of the National Institutes of Child Health and
Human Development and maintained by the Department of Biological
Sciences, University of Iowa, Iowa City, IA. This antibody
predominantly stains extracellular type I procollagen. The M38 antibody
was obtained from Takara Biomedicals, Shiga, Japan. This antibody
stains cellular and extracellular type I procollagen. Stained sections
were examined by light microscopy. The amount of staining (cellular and
extracellular) with M38 was assessed visually and scored as 0, 0.25,
0.5, 0.75, or 1, where 0 indicates no staining and 1 indicates
extensive extracellular staining as well as staining of most of the
interstitial cells. The three other values (0.25, 0.5, and 0.75)
represent intermediates between these two extremes.
Quantitative Fibroblast Outgrowth Assay
Skin samples were cut into small fragments (12 to 15 fragments per 4-mm biopsy) and each fragment placed in a separate well of a 96-well plate. Tissue fragments were incubated for up to 1 month in Dulbecco’s modified minimal essential medium of Eagle with nonessential amino acids and 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. The number of tissue fragments that yielded fibroblasts was determined at the end of the incubation period24 and expressed as a percentage of the total number of tissue fragments incubated. Cells were defined as fibroblasts on the basis of spindle-shaped morphology, reactivity with antibodies to vimentin, and a lack of reactivity with antibodies to keratin. Fibroblasts isolated in this manner were used without subculture or passaged 1 to 2 times before use.
Assessment of Type I Procollagen Synthesis and Fibroblast Proliferation in Vitro
Fibroblasts cultured from photodamaged forearm and sun-protected hip skin were plated in Dulbecco’s modified minimal essential medium of Eagle with nonessential amino acids and 10% fetal bovine serum at 8 x 104 cells per well in a 24-well culture plate. After allowing the cells to attach and spread, cells were washed twice in MCDB-153 basal medium (Clonetics Inc., Walkersville, MD), supplemented with 1.4 mmol/L Ca2+ (final concentration) and incubated for 2 days at 37°C and 5% CO2. At the end of the 2-day incubation period, cells were washed twice in Ca2+-supplemented MCDB-153 and incubated for an additional 1 hour at 37°C and 5% CO2. The 1-hour culture fluid was collected and analyzed for type I procollagen protein by enzyme-linked immunoassay (Takara Biomedicals). Preliminary studies showed that the rate of accumulation of immunoreactive type I procollagen in medium conditioned by 8 x 104 dermal fibroblasts was linear through at least 2 hours. After collection of the culture medium, cells were harvested by brief exposure to trypsin/ethylenediaminetetraacetic acid (EDTA) and counted with the aid of a particle counter.
Preparation of Polymerized Collagen Gels
Rat tail collagen (4.7 mg/ml in 1 N HCl; Collaborative Biomedical Products, Bedford, MA) was diluted to 1 mg/ml with Ca2+-supplemented MCDB-153. The solution was made isotonic by addition of an appropriate amount of a 10x concentrated solution of Hanks’ balanced salt solution, and the pH brought to 7.2. The collagen solution was added to wells of a 24-well plate (0.5 ml/well) and incubated for 2 hours at 37°C. During this period, the collagen formed a polymerized gel.25,26
Collagen-Degrading Enzyme Preparations
A collagenolytic enzyme preparation from Clostridium
histolyticum (Collagenase type I; Worthington Biochemical Corp,
Freehold, NJ) was used to produce fragmentation of the collagen. This
enzyme preparation contains collagenolytic activities at 105 and 55
kd,27
and the presence of these activities was confirmed
by reactivity with gelatin and monomeric collagen, but not with
ß-casein in zymography. Reactivity was lost when 10 mmol/L of EDTA
was included in the overnight incubation buffer. The bacterial enzyme
preparation cleaves intact collagen at numerous sites to produce low
molecular weight fragments.27
Collagenolytic activity was
quantified by exposing 1 mg of rat tail (monomeric) collagen to varying
concentrations of enzyme preparation for 5 hours at 37°C. Intact
collagen exposed to buffer alone served as control. At the end of the
incubation period, the control collagen and enzyme-treated collagen
were resolved on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis and stained with Coomassie brilliant blue. Laser
densitometry was used to quantify 1(I) and 2(I) bands in the
intact and digested preparations. When 10 mmol/L of EDTA was included
in the reaction mixture, no detectable collagen breakdown occurred.
Human basal cell carcinoma tissue was used as a source of collagen-degrading enzymes from human skin. Fresh tumor specimens obtained at surgery were cut into 2-mm pieces, and 6 to 8 tissue pieces incubated for 72 hours in 0.5 ml of Ca2+-supplemented MCDB-153. Incubation was at 37°C and 5% CO2. At the end of the incubation period, the culture fluid was obtained and used as the enzyme source. The conditioned medium from basal cell tumors contains large amounts of active MMP-1 as well as small amounts of MMP-8 (neutrophil collagenase) and MMP-13 (collagenase-3).28 Active forms of gelatinolytic enzymes (eg, MMP-2 and MMP-9) are also present.28 Zymography with gelatin, collagen, and ß-casein was used in the present study to confirm the presence of these activities, and collagen-degrading activity was quantified using digestion of monomeric collagen followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolution as described above. As with the bacterial enzyme preparation, inclusion of 10 mmol/L of EDTA in the incubation buffer suppressed zymographic activities, and inclusion of 10 mmol/L of EDTA in the reaction mixture suppressed collagen degradation.
Polymerized collagen gels were treated for 5 hours at 37°C with varying amounts of either the bacterial enzyme or human-skin enzyme preparation. At the end of the incubation period, the collagenase solutions were decanted. The polymerized collagen gels were briefly exposed in sequence to 10 mmol/L of EDTA and 14 mmol/L of Ca2+, and then rinsed exhaustively with Ca2+-supplemented MCDB-153.
Assessment of Collagen Contraction and Type I Procollagen Synthesis by Fibroblasts on Polymerized Collagen Gels
Four isolates of adult fibroblasts (two from forearm and two from hip) and five isolates of neonatal foreskin fibroblasts at passage 1 to 2 were added to the collagen gels at a final concentration of 1 to 8 x 104 cells per culture. For this, Ca2+-containing MCDB-153 medium was further supplemented with 0.1 ng/ml of epidermal growth factor, 0.5 µg/ml of insulin, and 2% of pituitary extract. Cells were incubated for 4 days, with fresh culture medium provided on day 2. Contraction of the collagen gels occurred during a 2-day period. The diameter of the collagen gel was measured at day 2 using a microscope with a calibrated grid in the eyepiece. Collagen contraction in this assay depends on fibroblasts binding to the collagen fibers and pulling the fibers as the cells themselves undergo actin-mediated and myosin-sliding filament-mediated contraction.29,30
At the end of the incubation period (day 4), the culture fluid was removed, and the collagen gels rinsed two times with Ca2+-supplemented MCDB-153 (without the added growth factors). Fresh culture medium (Ca2+-supplemented MCDB-153 without growth factors) was added to the wells and incubated for a further 1 hour. The 1-hour culture fluid was collected and assayed for type I procollagen by enzyme-linked immunosorbent assay as described above. The cells were then released from the collagen gels by sequential treatment with a high concentration of the bacterial collagenase preparation (100 µg for 2 hours) and trypsin (0.5% for 15 minutes) and counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Damage to the collagenous matrix of the dermis has been observed
at both the light- and electron-microscopic levels in photoaged
skin.31-37
Reductions in both the number and size of the
collagen fiber bundles as well as ultrastructural abnormalities in the
collagen fibrils themselves have been noted. However, the presence of
elastotic material often masks structural evidence of damage, and makes
quantification of damage difficult. In the present study we used
transmission electron microscopy in conjunction with a sensitive
(albeit, indirect) biochemical assay to compare structural features of
the collagen in severely photodamaged skin and in matched sun-protected
skin from the same individuals. Consistent with past
reports,32-34,37
large bundles of collagenous fibers were
present throughout the dermis of sun-protected skin. Healthy
fibroblasts in intimate contact with the collagen bundles could be seen
(Figure 1, A and C)
. In contrast,
severely photodamaged skin was characterized by the presence of fewer
bundles of collagen, and many individual, disorganized fibers. The
space between the collagen bundles, where not occupied with elastotic
material, was filled with mostly acellular debris. Instead of being in
contact with intact collagen, many of the fibroblasts in the damaged
skin were surrounded by the debris. Some of the cells demonstrated a
rounded rather than elongated morphology and, in some cases, there were
aggregates of two or more cells. These features are shown in Figure 1, B and D
. Thus, electron microscopy proved useful for identifying a
reduction in the relative amount of intact collagen in the photodamaged
skin, the presence of acellular debris, and contact/interaction of
dermal fibroblasts with this debris rather than with intact collagen.
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1,500 A) and
periodicity in photoaged skin as in sun-protected skin, others appeared
shortened and thinned. To quantitatively assess collagen fragmentation,
we took advantage of the fact that intact collagen is insensitive to
in vitro hydrolysis by -chymotrypsin, whereas collagen
that has been partially degraded in vivo is susceptible to
further hydrolysis by this enzyme in vitro.21
Digestion of partially degraded collagen by -chymotrypsin liberates
collagen fragments from the tissue, and the liberated collagen
fragments can be quantified by hydroxyproline
measurement.22
Hydroxyproline content after
-chymotrypsin digestion is, therefore, a measure of
partially degraded collagen in the tissue. Figure 2
compares amounts of hydroxyproline
released by -chymotrypsin treatment of matched samples of severely
photodamaged forearm skin and sun-protected hip skin from nine
individuals. The amount released from photodamaged skin was 3.6-fold
higher than the amount released from matched sun-protected skin.
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We next assessed levels of type I procollagen (1) gene
expression and type I procollagen (1) protein expression in severely
photodamaged forearm and sun-protected hip skin. As shown in Figure 3
, type I procollagen (1)
mRNA-expressing cells were readily detected in the dermal connective
tissue of sun-protected hip skin. mRNA-expressing cells were observed
throughout the entire dermis. In severely photodamaged forearm skin
from the same individuals, cellular expression of type I procollagen
(1) mRNA was markedly reduced (Figure 3)
. The number of cells
expressing type I procollagen (1) mRNA was reduced by 65% in
severely photodamaged forearm skin, compared to sun-protected hip skin
(n = 7).
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1) revealed a pattern
of type I procollagen expression similar to that observed with in
situ hybridization. Using antibody M38, protein expression was
observed within cells and extracellularly throughout the dermis in
sun-protected hip skin (Figure 4)
. In
contrast, cellular and extracellular expression of type I procollagen
protein was nearly absent in severely damaged forearm skin (Figure 4)
.
Semiquantitative scoring for both cellular and extracellular staining
(as described in Materials and Methods) revealed a decrease in staining
of 57% in severely photodamaged skin, compared to sun-protected hip
skin (n = 9). When a second antibody (SP1.D8)
was used in place of M38, cellular bodies did not stain well but
extracellular type I procollagen did. The extracellular staining
pattern with this antibody was virtually identical to that seen with
M38 (not shown). Together, the reduced level of type I (1)
procollagen gene expression and reduced level of cellular type I
procollagen protein expression indicate that type I procollagen
synthesis is decreased in photodamaged skin relative to sun-protected
skin. The reduced procollagen synthesis in photodamaged skin is
unlikely to reflect the presence of fewer interstitial fibroblasts in
the sun-damaged skin. Past studies have reported no change or an
increased number of interstitial cells present in photodamaged
skin.38
Likewise, the ability of topical retinoid
treatment to induce type I procollagen synthesis in photodamaged skin
implies the existence of cells with potential for collagen synthesis in
the tissue.9,10
Finally, the present studies (see below)
show that equivalent numbers of fibroblasts can be isolated from
severely sun-damaged forearm skin and matched sun-protected hip skin.
Based on these data, we favor the interpretation that interstitial
fibroblasts are present in photodamaged skin but are producing little
type I procollagen.
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We next determined if reduced type I procollagen synthesis in vivo resulted from a permanent incapacitation of collagen synthetic activity in fibroblasts from photodamaged skin. To do this, we isolated dermal fibroblasts from fragments of severely photodamaged skin and from matched sun-protected skin. The frequency of fibroblast outgrowth was similar for the two skin sites. A total of 36 fibroblast isolates were obtained from 108 fragments of photodamaged skin (33%) and 43 isolates were obtained from 122 fragments of sun-protected hip skin (35%) (not statistically different based on chi-square test).
Fibroblasts isolated from severely photodamaged forearm skin and
corresponding sun-protected hip skin were examined for proliferation
in vitro in a 2-day assay. As shown in Figure 5A
, cell growth was virtually
indistinguishable between fibroblast isolates from the two skin sites.
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Having demonstrated that growth potential of fibroblasts from
photodamaged and sun-protected skin was similar, we next determined the
capacity of these cells to synthesize type I procollagen. As shown in
Figure 5B
, type I procollagen protein production was virtually
indistinguishable between fibroblast isolates from photodamaged and
sun-protected skin.
Type I Procollagen Production in Vitro Is Inhibited on Partially Degraded Collagen
Polymerized collagen gels were prepared as described in Materials
and Methods and treated with bacterial collagenase or with human skin
collagenase from conditioned medium of basal cell carcinomas. Enzyme
concentrations were standardized based on degradation of monomeric
collagen, as shown in Figure 6
. Both
enzyme preparations produced dose-dependent degradation of intact
collagen. The major difference between the two enzymes was the direct
formation of low molecular weight fragments by the bacterial enzyme and
the initial formation of three-quarter-size and one-quarter-size
fragments by the human skin collagenase, followed by subsequent
degradation of these fragments (Figure 6)
.
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, which shows a vertical section
through the plane of the collagen surface. On partially degraded
collagen, dermal fibroblasts initially spread on the surface.
Throughout the subsequent 1 to 2 day period, extensive cell-cell
contacts were formed. Cell-cell aggregation occurred, and as it did,
the collagen contracted around the aggregated cells. This can be seen
in Figure 7B
, which is a section through the three-dimensional cell
aggregate produced as the cells contracted the collagen around them.
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. In both cases,
collagen fibers of similar length and width were seen. As compared to
the collagen in whole skin, the reconstituted fibers were shorter;
periodicity was evident. The density of fibers was high in the collagen
gels not exposed to collagenolytic enzymes. Both vertical and
horizontal orientation of fibers was evident, and fibroblasts with
their numerous processes were in close and frequent contact with these
fibers. In contrast, the density of intact collagen fibers was much
lower in the gels that had been exposed to the collagenolytic enzymes.
Many of the fibers appeared shortened and fragmented. There was no
discernable orientation to the fibers in these gels, and a large amount
of debris was evident. Fibroblasts were surrounded by the debris, and
many cells showed decreased process formation. Overall, there were few
cell contacts with intact collagen fibers. Thus, the
interactions of dermal fibroblasts with reconstituted intact or
partially degraded collagen in vitro paralleled what was
observed in sun-protected versus severely sun-damaged skin
in vivo.
Figure 8
demonstrates that collagen
contraction requires both collagen digestion and fibroblast activity.
Figure 8A
shows the relationship between enzyme concentrations needed
to produce collagen fragmentation (see Figure 6
) and concentrations
that facilitated collagen contraction. Inhibitor studies indicated that
MMPs in the enzyme preparations are, in fact, responsible for collagen
digestion. When collagen gels were exposed to either enzyme preparation
in the presence of 10 mmol/L of EDTA, and then subsequently exposed to
fibroblasts (after neutralization of the EDTA with
Ca2+), no contraction of the collagen occurred
(Figure 8B)
. Other gels were treated with 10 µg of human recombinant
tissue inhibitor of metalloproteinase-2 (TIMP-2) or 10 µg of
aprotinin along with the human tumor enzyme preparation. Collagen
contraction was inhibited by TIMP-2 but no inhibition was observed in
the presence of aprotinin (Figure 8B)
. Figure 8C
demonstrates that
contraction of partially digested collagen was dependent on fibroblast
activity. Full contraction occurred in the presence of 4 to 8 x
104
cells and partial contraction was observed
with as few as 2 x 104
cells. However,
contraction did not occur when fewer cells (1 x
104) were used.
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. Reduced cell growth and type I procollagen elaboration were
observed with all of the neonatal and adult dermal fibroblast isolates
tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mechanisms underlying decreased collagen synthesis by fibroblasts in severely photodamaged skin are not completely understood. Based on the results of the present study, we conclude that whereas fibroblast synthesis of type I procollagen is greatly diminished in photoaged human skin in vivo, growth capacity and synthesis of type I procollagen by fibroblasts from sun-damaged skin and age-matched sun-protected skin are indistinguishable when the cells are removed from the skin and examined in vitro. It should be noted that whereas the studies described here made use of sun-exposed forearm skin and sun-protected hip skin for most of the comparisons, we did, in fact, have the opportunity to assess a number of parameters (ie, collagen fragmentation in vivo and fibroblast isolation rates, growth rates and type I procollagen production in vitro) in underarm skin from 18 of the same volunteers. Sun-protected underarm skin and sun-protected hip skin from these individuals were similar in regard to all of the parameters assessed.
Because our data indicate that equivalent numbers of fibroblasts can be isolated from photodamaged skin and sun-protected skin, and because our data are based on results of multiple isolates from both tissue sites (from nine different individuals), it is unlikely that the in vitro data are skewed by a small subpopulation of cells in the photodamaged skin that grow out from the tissue and demonstrate the same phenotype as fibroblasts from sun-protected skin in vitro. Rather, these studies indicate that reduced procollagen production observed in vivo in severely sun-damaged skin is not because of reduced synthetic capacity of the fibroblasts per se. Consistent with these observations, it has been demonstrated previously that synthesis of collagen (as well as fibronectin) is low or undetectable in organ cultures of sun-exposed skin relative to organ cultures of healthy young skin.24,39 Synthesis of both matrix components is normalized24,39 when the organ cultures are treated with concentrations of all-trans retinoic acid that induce collagen expression in photoaged skin in vivo.23 Taken together with these previous observations, the present finding that fibroblasts in severely photoaged skin are not intrinsically damaged (with respect to collagen production) provides a rationale for therapeutic intervention with agents such as all-trans retinoic acid to stimulate collagen synthesis to repair photodamaged skin.40,41 In a like manner, any number of other agents may be found that have the capacity to restore collagen synthetic capacity to dermal fibroblasts that have been switched off in some manner by the presence of severely damaged connective tissue. It should be noted that the present data, particularly the mRNA data, strongly argue for reduced procollagen synthesis (independent of changes in collagen degradation). None-the-less, altered (ie, increased) elaboration of matrix-degrading MMPs could also lead to reduced procollagen deposition in the extracellular matrix. In fact, we assessed a number of forearm and hip skin samples for collagenase levels, but no significant differences were noted (J Varani and SC Datta, unpublished observation). This is in contrast to findings from studies with acute UV-irradiated skin, where increased collagenase was observed relative to nonirradiated controls.7,8 This is also in contrast to findings in natural aging, where a higher level of collagenase was observed in sun-protected skin from >80-year-old individuals than in sun-protected skin of younger (18- to 29-year-old) individuals.42
Because dermal fibroblasts do not seem to be intrinsically damaged in severely photoaged skin, it follows that inhibitory influences within the in vivo environment of severely photodamaged skin may act in some way to prevent cells that are inherently capable of elaborating collagen from doing so. In vitro studies performed with intact and partially degraded collagen gels support this suggestion. When skin fibroblasts (either neonatal or adult) were added to polymerized collagen, they rapidly attached and spread; they continued to proliferate and synthesize type I procollagen. In contrast, when fibroblasts were added to collagen gels that had been exposed to collagenase, cell growth and type I procollagen synthesis were reduced. Although extrapolating from in vitro experiments to what may occur in vivo is difficult, these data provide evidence that fibroblast functions that are important for maintenance of dermal connective tissue are inhibited in the presence of fragmented collagen. It should be noted that whereas both cell growth and type I procollagen production were reduced on the degraded collagen, the decrease in procollagen production was greater. Whether this reflects a specific inhibition of procollagen synthesis or whether procollagen elaboration is simply a more sensitive indicator of the overall functioning of the cells cannot be distinguished from the present data.
How damaged collagen exerts its influence on dermal fibroblast function is not known. A number of potential mechanisms exist. One possibility involves a change in cell shape that occurs during collagen contraction. Fibroblast interaction with structural collagen depends on the primary, secondary, and tertiary structure of the collagen fibrils.11 Fibroblasts attach to collagen fibers and express the typical, elongated spindle-cell morphology. When enough breaks are introduced into the three-dimensional collagen scaffold, it is no longer capable of resisting the contractile force of the cells and collapses. As the collagen scaffold collapses, the cytoskeleton disassembles and cell shape changes from elongated to round. Previous studies have demonstrated that optimal fibroblast function (including growth and collagen production) depend on maintenance of the elongated cell shape.43-45 Thus, it may be that loss of cell shape in the presence of degraded collagen directly underlies reduced growth and collagen production on the partially degraded collagen gels. Based on the in vivo ultrastructural findings presented here, we suggest that loss of cell shape could underlie reduced collagen synthesis in severely photodamaged skin.
Alternatively, it may not be loss of cell shape per se that underlies
reduced growth and collagen production. Rather, it may be abnormal
signaling, brought about by cellular interactions with degraded
collagen rather than with the intact triple helical molecule, that
directly causes abnormal cell function. Cellular interactions with
collagen are mediated by multiple members of the ß1 integrin family,
including 1, 2, and 3.12-16,46
Although a number
of different subunits mediate cell adhesion to collagen, other
functions including collagen synthesis, elaboration of matrix-degrading
enzymes, and collagen contraction are mediated more specifically by
different subunits.12,13,15,16,46
When the collagen
fibers are degraded, the contribution of different integrins to
cell-matrix interactions changes,20
leading to alterations
in motility and, perhaps, alterations in proliferation/matrix
production. In support of this, Gardner and colleagues15
have argued that fibroblast interactions with collagen through specific
integrins rather than contraction of the collagen is directly
responsible for modulating collagen production because reduced collagen
synthesis is seen in the dermis of mice with a specific integrin gene
defect. It should be noted that although signaling through integrin
receptors has been well studied, these molecules are probably not the
only molecules through which cell-matrix interactions occur. Recent
studies have shown, for example, that cell interactions with collagen
can occur through discoidin domain receptors.47
A role for
these receptors in collagen metabolism has been suggested. Which cell
surface receptors are ultimately responsible for the modulation of cell
behavior in the presence of degraded collagen fibers will need to be
addressed experimentally in future studies.
Ultimately, it may not be possible to completely distinguish between altered cell shape because of contraction of the collagen versus altered signaling because of cell interactions with collagen fragments as the reason for decreased cell growth and new collagen synthesis. It has recently been shown that collagen fragments actively promote disassembly of focal adhesion contacts, resulting in cleavage of cytoskeletal proteins and loss of cell shape.19
Finally, it must be noted that although our studies have focused mainly on type I collagen (because it is by far the most abundant structural protein in the skin), the dermis contains a number of collagenous and noncollagenous extracellular matrix molecules. Virtually every one of these molecules can be degraded by members of the MMP family.48 Fibroblast interactions with enzyme-degraded forms of any of these molecules could result in faulty signaling and subsequent alterations in fibroblast function. By focusing on collagenous components in the present study, we do not mean to rule out possible contributions of other components of the extracellular matrix. Regardless of the molecular mechanisms underlying reduced collagen synthesis in photodamaged skin, the beneficial effects of agents such as all-trans retinoic acid may derive not only from direct action on fibroblasts to stimulate collagen synthesis9,10 and decrease collagenase expression,7,8 but also from indirectly promoting (through the newly synthesized collagen) additional matrix-regenerative signals not present in untreated photodamaged skin.
A final question concerns the possible relationship between fibroblast-induced contraction of partially degraded collagen and the structural features seen in severely photodamaged skin. Until now, we have considered collagen contraction only as an indicator of collagen damage or as a modulator of new collagen synthesis. Could the presence of extensive amounts of contracted collagen contribute directly to the clinical appearance of severely photodamaged skin? Although there is no direct evidence to show that contraction of damaged collagen contributes to coarse wrinkling in photoaged skin, collagen contraction in the context of repeated cycles of damage and repair could distort and disrupt structural features of the tissue.46 In support of such a possibility, it has been shown in a past study that skin wounds with minimal collagen damage (for example freeze wounds) heal without contraction of the collagen at the wound site and consequently, the healed skin at these sites is smooth. In contrast, where damage to the matrix is more extensive (in burn wounds or traumatic injury), collagen contraction occurs during wound closure. Healed skin at these sites is rough and wrinkled.49 Studies to determine whether collagen contraction could play a similar role in photoaged skin are in progress.
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Acknowledgements |
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Footnotes |
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Supported in part by a grant from Johnson and Johnson, and by grant CA 60958 from the United States Public Health Service.
Accepted for publication December 4, 2000.
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References |
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