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Decreased collagen production in chronologically aged skin: Roles of age-dependent alteration in fibroblast function and defective mechanical stimulation

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Reduced synthesis of collagen types I and III is characteristic of chronologically aged skin. The present report provides evidence that both cellular fibroblast aging and defective mechanical stimulation in the aged tissue contribute to reduced collagen synthesis. The reduction in collagen synthesis due to fibroblast aging was demonstrated by a lower in vitro production of type I procollagen by dermal fibroblasts isolated from skin of young (18 to 29 years) versus old (80+ years) individuals (82 +/- 16 versus 56 +/- 8 ng/ml; P < 0.05). A reduction in mechanical stimulation in chronologically aged skin was inferred from morphological, ultrastructural, and fluorescence microscopic studies. These studies, comparing dermal sections from young and old individuals, demonstrated a greater percentage of the cell surface attached to collagen fibers (78 +/- 6 versus 58 +/- 8%; P < 0.01) and more extensive cell spreading (1.0 +/- 0.3 vs. 0.5 +/- 0.3; P < 0.05) in young skin compared with old skin. These features are consistent with a lower level of mechanical stimulation on the cells in old versus young skin. Based on the findings presented here, we conclude that reduced collagen synthesis in chronologically aged skin reflects at least two different underlying mechanisms: cellular fibroblast aging and a lower level of mechanical stimulation.
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Cell Injury, Repair, Aging and Apoptosis
Decreased Collagen Production in Chronologically
Aged Skin
Roles of Age-Dependent Alteration in Fibroblast Function and
Defective Mechanical Stimulation
James Varani,* Michael K. Dame,* Laure Rittie,
Suzanne E.G. Fligiel,* Sewon Kang,
Gary J. Fisher,
and John J. Voorhees
From the Departments of Pathology *and Dermatology,
The
University of Michigan, Ann Arbor, Michigan
Reduced synthesis of collagen types I and III is char-
acteristic of chronologically aged skin. The present
report provides evidence that both cellular fibroblast
aging and defective mechanical stimulation in the
aged tissue contribute to reduced collagen synthesis.
The reduction in collagen synthesis due to fibroblast
aging was demonstrated by a lower in vitro produc-
tion of type I procollagen by dermal fibroblasts iso-
lated from skin of young (18 to 29 years) versus old
(80years) individuals (82 16 versus 56 8 ng/ml;
P<0.05). A reduction in mechanical stimulation in
chronologically aged skin was inferred from morpho-
logical, ultrastructural, and fluorescence microscopic
studies. These studies, comparing dermal sections
from young and old individuals, demonstrated a
greater percentage of the cell surface attached to col-
lagen fibers (78 6 versus 58 8%; P<0.01) and
more extensive cell spreading (1.0 0.3 vs. 0.5 0.3;
P<0.05) in young skin compared with old skin.
These features are consistent with a lower level of
mechanical stimulation on the cells in old versus
young skin. Based on the findings presented here, we
conclude that reduced collagen synthesis in chrono-
logically aged skin reflects at least two different un-
derlying mechanisms: cellular fibroblast aging and a
lower level of mechanical stimulation. (Am J Pathol
2006, 168:1861–1868; DOI: 10.2353/ajpath.2006.051302)
Reduction of fibrillar (types I and III) collagen is a char-
acteristic feature of chronologically aged skin and is en-
hanced in photodamage. This has been well described
using histological and ultrastructural approaches in the
past,
1–5
and our own recent studies have documented
this biochemically in both chronological aging
6
and pho-
toaging.
7
Collagen-degrading matrix metalloproteinases
(MMPs) are up-regulated in skin by UV radiation.
8,9
Re-
peated induction of these enzymes by exposure to solar
radiation over years or decades is likely responsible for
producing collagen fragmentation in sun-damaged skin.
During natural or chronological aging of the skin, the
same MMPs that are up-regulated acutely in response to
UV radiation are gradually increased. This has been ob-
served in monolayer culture with cells obtained from old
versus young subjects,
10–14
and our own studies have
shown gradual up-regulation of MMPs in intact (old ver-
sus young) skin.
15
Although destruction of existing collagen is, undoubt-
edly, central to the deleterious changes observed in
aged/photoaged skin, failure to replace damaged colla-
gen with newly synthesized material is also critical to the
overall pathophysiology. There is a sustained down-reg-
ulation in collagen synthesis in photodamaged skin rela-
tive to what occurs in healthy sun-protected skin
16
and in
chronologically aged, sun-protected skin compared with
what is seen in young skin.
15
Mechanisms underlying the loss of collagen synthesis
in photodamaged skin and chronologically aged skin
have not been fully delineated. In a recent series of
studies, we demonstrated that in severely photodam-
aged skin, the presence of fragmented collagen in the
dermis inhibited collagen synthesis. Based on results
from a variety of in vitro and in vivo approaches, we
Supported in part by United States Public Health Service grants DK59169
and AG013964.
Accepted for publication March 13, 2006.
Address reprint requests to James Varani, Ph.D., Department of Pa-
thology, The University of Michigan, 1301 Catherine Rd./Box 0602, Ann
Arbor, MI 48109 USA. E-mail: varani@umich.edu.
American Journal of Pathology, Vol. 168, No. 6, June 2006
Copyright © American Society for Investigative Pathology
DOI: 10.2353/ajpath.2006.051302
1861
concluded that damaged collagen did not support a level
of mechanical tension on resident fibroblasts necessary
for efficient collagen synthesis.
6,7,17,18
Whether a similar
mechanism contributes to decreased collagen produc-
tion in chronologically aged skin, and if so, to what extent
it is responsible for the overall collagen reduction ob-
served in aged skin, is not known. These issues are
addressed in the present study.
Materials and Methods
Skin Biopsies
For this study, individuals between the ages of 18 and 29
years (young cohort) and individuals 80 years or older
(old cohort) were recruited. Replicate 2- and/or 4-mm
punch biopsies of sun-protected hip skin were obtained
from each individual. The 4-mm punches were used for
fluorescence microscopic and ultrastructural analysis
and for assessment of type I procollagen levels. On ar-
rival in the laboratory, one of the 4-mm biopsies was
immediately frozen in optimal cutting temperature me-
dium (OCT), and the other piece was fixed in glutaralde-
hyde. The 2-mm punch biopsies were used for routine
histology at the light microscopic level and as a tissue
source for the isolation of dermal fibroblasts. For routine
histology, the biopsies were fixed in 10% buffered forma-
lin. Fibroblast isolation was accomplished as indicated
below. All procedures involving human subjects were
approved by the Institutional Review Board, and biopsies
were obtained after receiving informed consent.
Human Dermal Fibroblasts in Monolayer Culture
Fibroblasts were isolated from skin biopsies as described
previously.
15
Briefly, the biopsy was minced with a scis-
sors and forceps, and tissue fragments were transferred
to individual wells of a 24-well dish. Dulbecco’s modified
minimal essential medium supplemented with nonessen-
tial amino acids and 10% fetal bovine serum (DMEM-
FBS) was used as culture medium. Only a minimal
amount of medium was included so that tissue pieces
would adhere to the plastic surface. The dishes were
maintained at 37°C in an atmosphere of 95% air and 5%
CO
2
. Eventually, fibroblast proliferation around the edge
of some of the tissue fragments was observed. When
cells in sufficient number were available, they were har-
vested with trypsin/ethylenediamine tetraacetic acid
(EDTA) and grown in monolayer culture using DMEM-
FBS. Cells were subcultured by exposure to trypsin/EDTA
one or two times and then used in experiments.
Type I Procollagen
Serial frozen sections were prepared from OCT-embed-
ded skin biopsies as follows: 7, 200, and 7
m. After
hematoxylin and eosin staining, the areas of the sections
from both ends of the 200-
m sample were measured
using Image ProPlus software to calculate the volume of
the 200-
m sample. Soluble protein extracts were pre-
pared from the 200-
m samples, which were homoge-
nized in ice-cold extraction buffer (50 mmol/L Tris-HCl,
pH 7.4, 0.15 mol/L NaCl, 1% Triton X-100, and protease
inhibitors [Complete Mini, Hoffmann-LaRoche, Nutley,
NJ]), and vortexed in the presence of glass beads (Bio-
spec, Bartlesville, OK). After centrifugation for 10 minutes
at 10,000 gand 4°C, supernatants were assayed for
procollagen I using a commercial enzyme-linked immu-
nosorbent assay kit (Panvera, Madison, WI) as described
previously.
15
The procollagen assay uses an antibody to
the C-terminal propeptide region that is part of the colla-
gen molecule as it is synthesized and secreted (before
being proteolytically cleaved). As such, this assay is a
measure of newly synthesized collagen. Type I procolla-
gen concentrations were normalized to the volume of
tissue used for the preparation of each sample.
Type I procollagen production was also assessed in
dermal fibroblasts in monolayer culture. Fibroblasts were
seeded at 4 10
4
cells per well in 24-well plates using
DMEM-FBS as culture medium. The cells were allowed to
attach overnight. The next day, they were washed and
then incubated in keratinocyte growth medium (Cambrex
Bioscience, Walkersville, MD) supplemented with 1.4
mmol/L Ca
2
. Keratinocyte growth medium is a serum-
free, low-Ca
2
modification of MCDB-153 medium sup-
plemented with epidermal growth factor (EGF), insulin,
hydrocortisone, and pituitary extract. Cell numbers were
determined 2 days later by releasing the cells with tryp-
sin/EDTA and enumerating them using a particle counter
(Coulter Electronics, Hialeah, FL). At the time of harvest,
the serum-free culture fluids were collected and as-
sessed for type I procollagen using the same enzyme-
linked immunosorbent assay procedure and then normal-
ized to cell number.
Immunostaining and Confocal Microscopy
A mouse monoclonal antibody to vinculin was obtained
from Chemicon (Temicula, CA). The primary antibody
was visualized with rabbit anti-mouse IgG antibody
bound to Alexa Fluor 488 (Invitrogen, Carlsbad, CA) and
further amplified with Alexa Fluor 488 goat anti-rabbit
IgG. Alexa Fluor 546-phalloidin (Invitrogen) was used as
a probe for actin. Nuclei were counterstained with the
nuclear dye 4,6-diamidino-2-phenylindole, dihydrochlo-
ride (DAPI) (Prolong Gold; Invitrogen). (Note that Alexa
Fluor 488 is spectrally similar to fluorescein, whereas
Alexa Fluor 546 is spectrally similar to rhodamine.)
OCT-embedded frozen skin biopsies from young
and old individuals were used for staining. Briefly, the
frozen tissue sections were fixed with 4% formalde-
hyde for 20 minutes. After fixation, the tissue sections
were washed twice with wash buffer (0.05% Tween 20
in Dulbecco’s phosphate-buffered saline), followed by
permeabilization with 0.1% Triton X-100 for 10 minutes.
Tissue sections were again washed and then exposed
to a blocking solution consisting of 1% bovine serum
albumin in Dulbecco’s phosphate-buffered saline for
30 minutes. Next, the sections were treated with anti-
vinculin antibody in blocking solution for 1 hour. After
1862 Varani et al
AJP June 2006, Vol. 168, No. 6
three subsequent washing steps (5 minutes each),
each sample was treated with Alexa Fluor 488-conju-
gated secondary antibody in blocking solution and
incubated for 45 minutes. Sections were simulta-
neously stained for actin expression using Alexa Fluor
546-phalloidin along with the secondary antibody. Af-
ter three additional washing steps (5 minutes each),
tissue sections were treated for 30 minutes with the
amplification antibody (Alexa Fluor 488 goat anti-rabbit
IgG). This was followed by two additional washing
steps and treatment with DAPI (nuclei counterstain) for
3 minutes. After three final washing steps, tissue sec-
tions were rinsed once with water. Coverslips were
mounted onto the microscope slides with Prolong Anti-
Fade (Invitrogen). Stained tissue sections were exam-
ined by fluorescence microscopy. Microscopy was
performed on a Zeiss LSM 510 confocal microscope
using a 63(C-Apochr) water immersion objective
lens (numerical aperture (NA) 1.2). Laser excitation
wavelengths included 364, 488, and 543 nm scanned
in sequence by the line method.
Light Microscopic and Transmission Electron
Microscopic Studies
Skin biopsies were fixed overnight in 2% glutaraldehyde
in 0.1 mmol/L cacodylate buffer (Sigma, St. Louis, MO) at
pH 7.4. Glutaraldehyde-fixed specimens were treated
with 2% osmium tetroxide buffered in 0.1 mmol/L caco-
dylate buffer. Specimens were dehydrated with graded
ethanol to 2 100% ethanol and 2propylene oxide
(EM Sciences). The samples were embedded in pure
epon resin. One-micrometer tissue sections were cut,
stained with toluidine blue, and examined at the light
microscopic level. Surface area occupied by individual
cells was assessed quantitatively using NIH Image soft-
ware on 1-
m sections obtained from glutaraldehyde-
fixed, plastic-embedded tissue.
The same tissue sections used for quantification of
surface area at the light microscopic level were also used
to identify areas of interest for transmission electron mi-
croscopy. Ultrathin sections were cut from areas of inter-
est, stained with lead citrate and uranyl acetate (both
from EM Sciences), and observed using a Phillips 400
transmission electron microscope. Photographs were
made from several areas of each specimen. Using the
high-resolution photographs, interstitial cells were quan-
titatively evaluated for percentage of the cell boundary in
contact with individual collagen fibrils or collagen fibril
bundles. Although it is difficult to identify interstitial fibro-
blasts with 100% accuracy, obvious contaminants (mast
cells, cells in vascular structures, glandular epithelial
cells, and red blood cells) were not evaluated.
Statistical Analysis
Proliferation, type I procollagen production, two-dimen-
sional surface area measurements and collagen-attach-
ment data were compared between young and old skin.
The data were analyzed using Student’s two-sample t-
test (Microsoft Excel and SAS analytic software). Sum-
mary data are expressed as means SEM. All Pvalues
are two-tailed.
Results
Type I Procollagen Synthesis in Skin from
Young and Old Individuals
In the first series of experiments, type I procollagen con-
tent was measured in skin from young and old individu-
als. Type I procollagen content, a marker of ongoing
collagen synthesis, was decreased by 68% in old skin
versus young skin (Figure 1).
Type I Procollagen Synthesis by Fibroblast
Isolates from Young and Old Skin
Next, conditioned medium from cultures of young and old
fibroblasts was assessed for type I procollagen produc-
tion. Isolates from the cohort of young individuals synthe-
sized more type I procollagen than did the fibroblast
isolates from the cohort of old individuals (Figure 2). Cells
isolated from young skin also proliferated to a greater
extent (Figure 2).
Collagen Structure, Fibroblast-Collagen
Interactions, and Cell Shape in Vivo:
Comparison of Young and Old Skin
A series of related histological, ultrastructural, and fluo-
rescence microscopic analyses were performed to de-
termine the relationship between collagen structure, cell
shape and adhesion site protein distribution. Differences
in fiber bundle content were observed between young
and old skin. Fiber bundles were thicker and there was
Figure 1. Type I procollagen production in young and old skin. Values
shown are averages SEM, based on six young and six old individuals.
Statistical significance of the differences between young and old skin was
determined using Student’s t-test. *Significance at the P0.05 level.
Collagen in Aged Skin 1863
AJP June 2006, Vol. 168, No. 6
less open space within and between bundles in the pap-
illary dermis of the 18- to 29-year-old individuals than in
the corresponding tissue from the 80-year-old subjects
(Figure 3). In sections of young skin, interstitial cells could
be seen oriented in the plane of the collagen polymer
(Figure 3, inset). In old skin, the papillary dermis was
characterized by the presence of open space inter-
spersed with criss-crossing, tangled, thin fibers. There
was little evidence of fiber bundle orientation. Open
space around interstitial cells was apparent (Figure 3B,
arrows), and there was little evidence of cell orientation
(Figure 3, inset).
Cell shape (two-dimensional cross-sectional area of
cells in 1-
m-thick sections from plastic embedded tis-
sue) was quantitatively examined. Cells from young indi-
viduals were more spread than the corresponding cells
from old individuals (Figure 4). The quantitative data
shown in Figure 4 were obtained from sections of super-
ficial (papillary) dermis. When the same analyses were
done in the deeper layers of the (reticular) dermis, results
were similar (not shown).
Ultrastructural features of the matrix were compared in
sections from young and old skin. Overall, there were no
major differences in the appearance of the collagen poly-
mers that could be used to distinguish young and old
skin. There were, however, areas in sections of old skin
where the density of collagen was reduced. Such areas
were primarily (though not always) confined to the pap-
illary dermis. Occasional unstriated fibers were present in
some of the sections from the old skin samples, but we
never detected the elastotic material characteristic of
badly sun-damaged skin.
2,3,5
As part of the analysis, the interaction between inter-
stitial cells and the surrounding collagen was examined.
For these studies, we avoided mast cells, epithelial cells
in glandular structures, cells associated with the micro-
vasculature and any inflammatory cell that might be
present. Thus, a majority of the cells characterized were
interstitial fibroblasts. Figure 5 demonstrates and quanti-
fies the interaction of these cells with the surrounding
collagen in sections from young and old individuals. In
skin sections from the 18- to 29-year-old cohort, cells
were in contact with intact collagen fibrils over a greater
proportion of their surface (two-dimensional image) than
were cells in sections from old skin.
Adhesion Site Protein Expression: Comparison
of Cells in Young and Old Skin
In a final set of experiments, anti-vinculin antibody was
used to identify focal adhesion sites by fluorescence
microscopy (Figure 6). In cells from both young and old
skin, anti-vinculin staining was evident. Staining in skin
sections from young individuals was associated with fiber
bundles, which were evident by their dull orange appear-
ance in the stained sections. In sections of old skin, there
were fewer focal adhesions, and much of the staining
was closely associated with nuclei (blue color). The dif-
ferential pattern of adhesion site protein expression
shown in Figure 6 correlated with differences in cell
spreading, as observed at the light microscopic level
(Figure 4), and with cell-collagen interactions, as ob-
served at the transmission electron microscopic level
(Figure 5). Alternatively, nuclear-associated fluorescence
could indicate an intracellular (presumably, functionally
inactive) pool of vinculin.
Discussion
There is a large body of literature demonstrating a rela-
tionship between mechanical tension on cells in vitro and
biological responses of cells to stress.
19–23
When there is
a sufficient level of mechanical tension on fibroblasts,
production of collagen and other components of the ex-
Figure 2. Cellular proliferation and type I procollagen production in mono-
layer culture. Proliferation (A) and type I procollagen production (B)by
fibroblasts isolated from young and old skin. Values shown are averages
SEM, based on 26 fibroblast isolates from eight young individuals and 37
isolates from eight old individuals. Statistical significance of the differences
between isolates from young and old skin was determined using Student’s
t-test. *Significance at the P0.05 level.
1864 Varani et al
AJP June 2006, Vol. 168, No. 6
tracellular matrix is high. When tension is reduced, matrix
production falls, and elaboration of matrix-degrading en-
zymes is concomitantly stimulated.
24–30
Lapiere and col-
leagues
31
directly measured mechanical forces gener-
ated by fibroblasts in three-dimensional collagen lattices.
In a series of studies, they demonstrated that the reduc-
tion in collagen synthesis occurring as a consequence of
reduced mechanical tension reflected decreased tran-
scription of genes for interstitial collagens, effects on
enzymes involved in the posttranslational processing of
procollagen peptides, and increased elaboration of col-
lagen-degrading MMPs. It was shown, furthermore, that
multiple signaling pathways were responsible for altered
gene transcription.
32,33
Of interest, changes in MMP pro-
duction resulting from a loss of mechanical tension could
be clearly separated from changes resulting from inter-
leukin-1 stimulation.
34
Consistent with these past observations, studies from
our laboratory demonstrated that when human dermal
fibroblasts were maintained on native three-dimensional
collagen lattices, these cells produced type I procolla-
gen. When the collagen in the lattice was fragmented by
exposure to MMP-1 (interstitial collagenase) or to en-
zymes elaborated by UV-exposed skin (primarily MMP-
1), a fall-off in collagen production occurred. Concomi-
tant with the decrease in collagen production on the
fragmented collagen lattices was a reduction in mechan-
ical tension as evidenced by reduced cell spreading,
decreased focal adhesions, and dissolution of actin
stress fibers.
6,7,17,18
Parallel studies showed that in se-
verely photodamaged skin (with its extensive collagen
degradation), resident fibroblasts were characterized by
similar morphological, ultrastructural, and fluorescence
microscopic findings as observed in vitro on fragmented
collagen.
18
Our interpretation of these findings is that
degradation of collagen in severe photodamage pro-
duces an environment that is unable to support a level of
mechanical tension required for efficient collagen-syn-
thetic activity.
Collagen fragmentation, a reduction in total collagen,
and decreased cell-collagen fiber interactions also char-
acterize chronologically aged skin.
1–3,5
The enzymes re-
sponsible for collagen degradation increase gradually
over time in the skin.
15
Collagen synthesis may also de-
cline gradually, but the fall-off in new collagen production
is most evident when skin damage is clinically evident.
15
The present study was undertaken to determine what
factors contribute to the loss of collagen-synthetic activity
in chronological aging. Based on the results presented
here, we suggest at least two mechanisms. In vitro stud-
ies indicate that reduced collagen synthesis in old skin
reflects, at least in part, an age-related reduction in col-
lagen-synthetic activity in the resident population of fibro-
blasts. Fibroblasts obtained from sun-protected skin of
young adults (18 to 29 years of age) synthesized an
average of 82 ng of type I procollagen per 5 10
4
cells,
whereas cells from old individuals (80years of age)
synthesized 56 ng per 5 10
4
cells under identical in
vitro conditions. Coupled with this is the fact that there are
fewer interstitial fibroblasts in aged skin compared with
young skin,
15
contributing to reduced growth capacity
(Figure 2). Thus, even when all environmental factors that
may contribute to differences in vivo are removed, there is
still an age-dependent difference in collagen-synthetic
capacity that explains at least part of the previously doc-
umented reduction in collagen content of aged skin.
6
If a decrease in collagen synthetic capacity occurs as
a function of fibroblast (cellular) aging, then what role
does a reduction in mechanical tension play? Although it
may be difficult to precisely estimate the percentage of
the overall decrease in collagen production (in old skin
relative to young skin) accounted for by decreased me-
chanical tension, the following analysis serves as a basis
for comparison. Our past studies
15
indicate that collagen
Figure 3. Histological features of sun-protected skin from young and old individuals as observed in 5-
m hematoxylin and eosin-stained sections from
formalin-fixed tissue (main frames) and in 1-
m toluidine blue-stained sections from glutaraldehyde-fixed, plastic-embedded tissue (insets). Thick fiber bundles
are present throughout the upper dermis of sun-protected young skin. Inset: Some fibroblasts can be seen oriented in the plane of the fiber bundles. In the old
skin sample, the bundles have been replaced with thin, disorganized fibers. There is more open space in the dermis. Interstitial cells are round or oblong, and
some are surrounded by open space (arrows). Inset: Fibroblast orientation (arrow) is not evident. Hematoxylin and eosin-stained sections, 490; toluidine
blue-stained sections (insets)980.
Collagen in Aged Skin 1865
AJP June 2006, Vol. 168, No. 6
production in sun-protected skin of old (80years) indi-
viduals is decreased by approximately 75% relative to
production in corresponding skin of young (18 to 29
years) adults. Both Western blotting of skin extracts and
immunostaining for type I procollagen are consistent in
this regard. These past findings are also consistent with
the direct measurements of type I procollagen in young
and old skin presented here (ie, 68% reduction in old
versus young skin; Figure 1). If the number of fibroblasts
in skin from 80-year-old individuals is reduced by ap-
proximately 35% relative to the number in skin of 18- to
29-year-old individuals as indicated by morphometric
analysis
15
and if type I procollagen synthesis is reduced
by an average of 30% in fibroblasts from old skin as
indicated in Figure 2 of the present study, then it is
reasonable to suggest that age-dependent differences in
fibroblast biosynthetic activity account for approximately
45% of the total decrease. Other factors (including loss of
mechanical tension) account for the remaining 30%. This
separation is shown schematically in Figure 7. How ulti-
mate accuracy of the 45%/30% split is less important
than the fact that while a loss of mechanical tension
appears to be the major factor underlying decreased
collagen synthesis in photodamaged skin,
18
in chrono-
logically aged skin, it is one of the two contributing
mechanisms.
Of interest, age-dependent alterations in fibroblast bio-
synthetic activity and the reduction in external mechani-
cal tension on cells in the dermis of aged skin may not be
independent. The same reduction in mechanical tension
that lowers collagen production by resident fibroblasts
may also indirectly contribute to permanent alterations in
fibroblast function. It is generally accepted that pheno-
typic changes seen in aged fibroblasts are largely medi-
ated by oxygen radical damage.
35
This is potentially
related to the issue at hand, because among the alter-
ations that occur under conditions of reduced mechani-
cal tension is increased oxidant stress as evidenced by
Figure 4. Shape of fibroblasts in the papillary dermis of sun-protected hip
skin from young and old individuals (1-
m toluidine blue-stained sections
from glutaraldehyde-fixed, plastic-embedded tissue). Top panel: Cells in
young skin are flattened, and cytoplasm and nucleus are visible (arrow).
Cells are embedded in matrix. Cells in old skin appear round, and only the
nucleus and a small amount of cytoplasm are visible (arrows). Bottom
panel: Surface area measurements were made quantitatively as described in
Materials and Methods. Values represent mean cross-sectional surface area
SEM, based on 160 cells in sun-protected skin from six young individuals and
57 cells in sun-protected skin from six old individuals. Statistical significance
was determined using Student’s t-test (two-tailed). *P0.01 (magnification,
240).
Figure 5. Ultrastructural appearance of dermal fibroblasts in healthy sun-
protected hip skin from young and old individuals. Aand B: The cell from the
section of young skin (A) is flattened and well spread. The cell is in contact
with collagen fibers over a high percentage of its surface. The cell in the old
skin sample (B) is round and is in contact with collagen polymer over a
smaller portion of its surface. There is more open space surrounding the cell.
(The computer-generated coloring of the cells was done to aid in the
demarcation of cells from extracellular material [magnification 2050]). C: A
high magnification (3500) of old skin showing the striations in the fibers
(typical of collagen). D: Quantification of contact between cells and collagen
fibers. Values represent the percentage of the cell boundary in contact with
collagen fibers SEM (P0.01; two-tailed Student’s t-test). Measurements
are based on 33 cells in sections of healthy skin from six young individuals
and 38 cells in sections from six old individuals.
1866 Varani et al
AJP June 2006, Vol. 168, No. 6
increased levels of reactive oxygen species and altered
expression of anti-oxidant enzymes (G.J. Fisher, unpub-
lished observations). It might be inferred from this that
environmental damage causally precedes changes in
fibroblast function that are observed in the aged or se-
nescent state.
The age-related decrease in collagen-synthetic activity
may be, at least in part, reversible. It has been demon-
strated that agents such as all-trans retinoic acid can
stimulate collagen production in aged skin.
16,36–38
Not
surprisingly, topical retinoid use brings about an im-
provement in the appearance of aged skin
39
and photo-
damaged skin.
40
In summary, collagen synthetic capacity is low in aged
(sun-protected) skin relative to that in healthy young sun-
protected skin. Based on the findings presented here
and by analogy with in vitro models and the findings of
photoaging studies, we hypothesize that old fibroblasts
have an age-dependent reduction in the capacity for
collagen synthesis and simultaneously experience a loss
of mechanical stimulation resulting from decreased intact
collagen fibers.
Acknowledgments
We thank Suzan Rehbine for help with recruitment of
volunteers, Robin Kunkel and Lisa Riggs for help with
electron microscopy, Ted Hamilton for help with statisti-
cal analysis, Bruce Donohoe for help with fluorescence
microscopy, and Laura Vangoor for help with the prepa-
ration of graphic materials.
Figure 6. Adhesion-site protein expression in sections of healthy sun-pro-
tected hip skin from young and old individuals. Tissue sections (OCT-
embedded frozen) were stained using antibody to vinculin and concomi-
tantly with phalloidin (actin stain) and DAPI (nuclear stain) as described in
Materials and Methods. After staining, cells were examined by confocal
fluorescence microscopy. Cells are identified by their blue (DAPI)-stained
nuclei. Bright green punctate fluorescence identifies vinculin. In the 18- to
29-year-old skin samples, vinculin can be seen at a distance from the nucleus,
and in many areas, the vinculin appears to be in close apposition to collagen
fibers. In the 80-year-old skin, blue-stained nuclei are apparent, but there
is less vinculin than in the young skin samples. Where intense focal staining
is evident, it is surrounding the nucleus (arrows). Away from the nucleus,
staining is more diffuse than seen in cells from young skin. The sections
presented are representative of young and old sun-protected skin from six
individuals, respectively. In both young and old skin, collagen fibers are
apparent by their dull orange fluorescence (magnification, 1200).
Figure 7. Schematic representation of mechanisms underlying reduced col-
lagen synthesis in aged skin.
Collagen in Aged Skin 1867
AJP June 2006, Vol. 168, No. 6
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1868 Varani et al
AJP June 2006, Vol. 168, No. 6
... It should be emphasized that changes in the organization and structure of the collagen matrix are characteristic of both chrono-and photoaging of human skin [2]. The results of the study of skin biopsy samples of elderly people have shown that the accumulation of degraded/fragmented fibers and a decrease in de novo collagen synthesis correlate both with age and with the degree of photo-damage severity [51][52][53][54]. ...
... When the CM integrity is damaged, which occurs both during chronological aging and photoaging, changes are observed in mechanotransduction (transmission of mechanical signals from ECM to cells), promoting development of the mechanism that disrupts DF functions ( Figure 2) [13,53,83,84]. ...
... It has been revealed that integrins (heterodimeric transmembrane proteins, specific receptors, primarily, α1β1 and α2β1 to collagen type I [85]) located on the cell surface are able to specifically bind ECM proteins, in particular type I collagen [53]. The adhesion of integrins to ECM proteins contributes to the formation of bonds not only between integrins and the collagen matrix but also with actin (protein of the DF cytoskeleton) since integrins are attached to the CM from the outer surface of the cell membrane and connected to the cytoskeleton from the inner surface of the cell membrane, thereby creating focal adhesion complexes (focal contacts) that ensure the closely related regulatory and mechanical functions of DFs [11,53,86]. ...
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Skin aging is a multi-factorial process that affects nearly every aspect of skin biology and function. With age, an impairment of structures, quality characteristics, and functions of the dermal extracellular matrix (ECM) occurs in the skin, which leads to disrupted functioning of dermal fibroblasts (DFs), the main cells supporting morphofunctional organization of the skin. The DF functioning directly depends on the state of the surrounding collagen matrix (CM). The intact collagen matrix ensures proper adhesion and mechanical tension in DFs, which allows these cells to maintain collagen homeostasis while ECM correctly regulates cellular processes. When the integrity of CM is destroyed, mechanotransduction is disrupted, which is accompanied by impairment of DF functioning and destruction of collagen homeostasis, thereby contributing to the progression of aging processes in skin tissues. This article considers in detail the processes of skin aging and associated changes in the skin layers, as well as the mechanisms of these processes at the molecular level.
... Indeed, the collagen V determines the diameter of the collagen I fibers with which it is associated, and the collagen IV forms the framework of the dermal-epidermal junction and other basal membranes. During aging, including intrinsic aging and extrinsic aging (induced by external stimuli), it has been shown that the quantity and quality of collagen fibers were diminished, associated with a loss of network density(18). Furthermore, collagen network is finely regulated during life. ...
Thesis
Hyaluronic acid (HA), a major glycosaminoglycan in the extracellular matrix, plays an important role in a number of biological processes such as wound healing, cancer or embryonic development. The catabolism of HA generates fragments that play a role in migration, apoptosis or immune regulation. HA metabolism is a finely regulated process, adapting its functions to its microenvironment. In the event of deregulation of the metabolism, the accumulation of HA fragments seems to be strongly related to the severity of chronic inflammatory pathologies. Nevertheless, due to its rheological and structural properties, HA prevents dehydration and maintains the structure of the skin. Although it is generally considered to be an inert and biocompatible material, rare cases of late side effects have been observed. Thus, in order to improve our understanding of the mechanisms that can lead to late complications, we hypothesized that the rare complications observed could be related to an inflammatory microenvironment. In this thesis work, we evaluated the inflammatory and vascular potential of degraded and non-degraded HA in dermal cells, immune cells and in a mouse model. In an inflammatory microenvironment, adding HA fragments to endothelial cells and dermal fibroblasts does not worsen the inflammatory response, unlike immune cells. This suggests the involvement of M1 macrophages and mature dendritic cells to a lesser extent in the transmission of inflammation to the tissue. In addition, in a mouse model of chronic low-grade inflammation we showed in vivo that injection of HA can lead to microvascular sensitivity in response to acetylcholine. The hypothesis is that this vascular sensitivity is linked to the recruitment of immune cells at the injection site. Furthermore, we demonstrated in vitro that in the absence of immune cells, an inflammatory vascular microenvironment could lead to an alteration of the extracellular matrix. These results show a diversity in cell-cell interactions depending on the inflammatory degree of the microenvironment, which in the presence of degradation fragments of HA (i.e. during aging or lesion) could weaken the skin tissue
... On this basis, human normal fibroblast was selected in this study for experiments to show the expression of angiogenesis and fibroblast proliferation genes FGF1 and FGF7, type I collagen genes COL1A1 and COL1A2, and type III collagen gene COL3A1. Fibroblasts produce substances that constitute the basic structure of skin; a decrease in their proliferation and activity causes the deterioration of the skin structure [18]. The activities of inhibiting α-MSH-stimulated melanogenesis and an inhibitory effect on MMP-1 secretion (HLE activates MMP-1) mean that it is expected that these activities and the two substances, α-EG and pinoresinol, will be useful for the prevention of photoaging and wrinkle formation, as well as collagen creation and skin whitening. ...
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α-EG is a unique substance that was first found in the leaves and fruits of Morinda citrifolia (Mc) growing in Thailand using GC-MS at 52.33% and 54.12%. It was then concentrated and its abundance quantified, along with that of pinoresinol, via GC, compared to the standards in leaves, ufp, rfp, rawfs, and seeds. α-EG and pinoresinol, which have collagen stimulating, skin whitening, and an inhibitory effect on wrinkle formation, were found in different concentrations and amounts. Three different concentrations of the five Mc part extracts were tested on NHDF for gene expression related to the aforementioned activities, COL1A1, COL1A2, and COL3A1, FGF1 and FGF7 by qRT-PCR. The results showed various expression levels, both stimulatory and inhibitory, with different concentrations of plant parts and genes. Similar results were revealed when the experiments were performed with Morus alba (Ma), which was found to contain 20.48 g protein p/100 g leaves at concentrations of 3.11 mg/mL. The studied Mc parts seem to have advantages based on the stated objectives, gene type and level of activity of each plant part. Rawfs and leaves supplemented with Ma samples were selected for toxicity tests with PBMCs. The lack of both cell and DNA toxicity from the rawfs indicated that they can be used safely.
... Previous studies have demonstrated that MMP-1, MMP-2, and MMP-3 are mainly MMPs for degrading collagens and elastin [7]. With the aging process of the skin, the collagen 2 of 14 biosynthesis decreases, and collagen fragmentation increases, which both contribute to an overall reduction in collagen content [8][9][10]. So, in recent years, many nutritional supplements and specialty foods containing collagen have been marketed to facilitate anti-aging of the skin. ...
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Skin aging is categorized as chronological aging and photo-aging that affected by intrinsic and extrinsic factors. The present study aimed to investigate the anti-aging ability and its underlying mechanism of chlorogenic acid (CGA) on human dermal fibroblasts (HDFs). In this study, CGA specifically up-regulated collagen I (Col1) mRNA and protein expressions and increased the collagen secretion in the supernatant of HDFs without affecting the cell viability, the latter was also demonstrated in BioMAP HDF3CGF system. Under ultraviolet A (UVA)-induced photoaging, CGA regulated collagen metabolism by increasing Col1 expression and decreasing matrix metalloproteinase 1 (MMP1) and MMP3 levels in UVA-irradiated HDFs. The activation of transforming growth factor-β (TGF-β)-mediated Smad2/3 molecules, which is crucial in Col1 synthesis, was suppressed by UVA irradiation and but enhanced at the presence of CGA. In addition, CGA reduced the accumulation of UVA-induced reactive oxygen species (ROS), attenuated the DNA damage and promoted cell repair, resulting in reducing the apoptosis of UVA-irradiated HDFs. In conclusion, our study, for the first time, demonstrate that CGA has protective effects during skin photoaging, especially triggered by UVA-irradiation, and provide rationales for further investigation of CGA being used to prevent or treat skin aging.
... Age-related increases in matrix metalloproteinases (MMPs) production are directly responsible for collagen degradation leading to a decline in collagen content at about 2% per year [14,15]. Dermal fibroblasts experience an alteration in the equilibrium between the synthesis of collagen and the synthesis of collagen-degrading enzymes under the influence of various stressors throughout life with further enhancement of dermal structural impairment [16]. Moreover, increased accumulation of degraded collagen fibers work as entrapment that manifests itself in the reduction of fibroblast functional quality and quantity; likewise, a decline in collagen and other ECM proteins renewal capacity is observed with age [15]. ...
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Modern understanding of aging is based on the accumulation of cellular damage during one's life span due to the gradual deterioration of regenerative mechanisms in response to the continuous effect of stress, lifestyle, and environmental factors, followed by increased morbidity and mortality. Simultaneously, the number of senescent cells accumulate exponentially as organisms age. Cell culture models are valuable tools to investigate the mechanisms of aging by inducing cellular senescence in stress-induced premature senescence (SIPS) models. Here, we explain the three-step and one-step H2O2-induced senescence models of SIPS designed and reproduced on different human dermal fibroblast cell lines (CCD-1064Sk, CCD-1135Sk, and BJ-5ta). In both SIPS models, it was evident that the fibroblasts developed similar aging characteristics as cells with replicative se-nescence. Among the most noticeable senescent biomarkers were increased β-Gal expression, high levels of the p21 protein, altered levels of cell-cycle regulators (i.e., CDK2 and c-Jun), compromised extracellular matrix (ECM) composition, reduced cellular viability, and delayed wound healing properties. Based on the significant increase in senescence biomarkers in fibroblast cultures, reduced functional activity, and metabolic dysfunction, the one-step senescence model was chosen as a feasible and reliable method for future testing of anti-aging compounds.
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The basal levels of mRNAs encoding two metalloproteinases, collagenase and stromelysin, were increased as a function of in vitro serial subcultivation (cellular aging) of human fibroblasts. Procollagenase and prostromelysin synthesis and secretion were also greater in the old cultures (late passage). In contrast, the steady-state expression of mRNA for an inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase-1 (TIMP-1), in late-passage cultures was lower than that in young cell cultures (early passage). Each mRNA was analyzed using total RNA preparations isolated from normal fibroblast cultures at different phases of the in vitro life span and from cultures derived from donors with the premature senescence syndromes characterized as Werner syndrome, progeria (Hutchinson-Gilford) syndrome, or Cockayne syndrome. In normal cell cultures expression of metalloproteinase mRNAs was increased after the culture had completed greater than 90% of the in vitro life span, and the reduction in TIMP-1 mRNA expression occurred after the culture had completed greater than 74% of the in vitro lifespan. In Werner syndrome cultures expression of metalloproteinase and TIMP-1 mRNAs was similar to the level of expression observed in late-passage cell cultures. Levels of metalloproteinase and TIMP-1 mRNA expression in progeria and Cockayne syndromes were similar to those of early-passage cell cultures. To determine if young and old cells were each responsive to mediators of metalloproteinase synthesis, cultures were treated with phorbol ester or cytokines. 12-O-tetradecanoylphorbol-13-acetate treatment increased the steady-state levels of all three mRNAs in young, old, and Werner syndrome cultures and increased procollagenase levels in all cultures. Early- and late-passage cell cultures also responded to cytokines. Interleukin-1 alpha treatment increased collagenase and stromelysin mRNA levels while transforming growth factor-beta reduced the steady-state levels of both transcripts. Neither cytokine affected the steady-state level of TIMP-1 mRNA. The results indicate that in vitro cellular aging is associated with changes in expression of mRNAs encoding proteins that mediate inflammatory responses and connective tissue remodeling.