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Rejuvenation of Gene Expression Pattern of
Aged Human Skin by Broadband Light Treatment:
A Pilot Study
Anne Lynn S. Chang
1
, Patrick H. Bitter Jr
2
,KunQu
1
, Meihong Lin
1
, Nicole A. Rapicavoli
1,3
and Howard Y. Chang
1,3
Studies in model organisms suggest that aged cells can be functionally rejuvenated, but whether this concept
applies to human skin is unclear. Here we apply 3
0
-end sequencing for expression quantification (‘‘3-seq’’) to
discover the gene expression program associated with human photoaging and intrins ic skin aging (collectively
termed ‘‘skin aging’’), and the impact of broadband light (BBL) treatment. We find that skin aging was associated
with a significantly altered ex pression level of 2,265 coding and noncoding RNAs, of which 1,293 became
‘‘rejuvenated’’ after BBL treatment; i.e., they became more similar to their expression level in youthful skin.
Rejuvenated genes (RGs) included several known key regulators of organismal longevity and their proximal
long noncoding RNAs. Skin aging is not associated with systematic changes in 3
0
-end mRNA processing. Hence,
BBL treatment can restore gene expression pattern of photoaged and intrinsically aged human skin to resemble
young skin. In addition, our data reveal, to our knowledge, a previously unreported set of targets that may lead
to new insights into the human skin aging process.
Journal of Investigative Dermatology (2013) 133, 394–402; doi:10.1038/jid.2012.287; published online 30 August 2012
INTROD UCTION
Aging is under complex genetic and environmental control.
Aging is associated with large-scale changes in gene
expression, and how such changes may be modulated for
healthful benefits in human beings is not clear. Numerous
single-gene mutations have been identified that can extend
the lifespan of model organisms (Partridge, 2010; de
Magalhaes et al., 2012), and dietary restriction can slow the
rate of aging, even if applied late in life (Partridge, 2010).
More recently, several interventions have been shown to
confer features of youthfulness to aged cells or tissues,
demonstrating a remarkable plasticity of the aging process.
For instance, heterochronic parabiosis between young and
old mice enables circulatory factors to restore the functions of
aged muscle stem cells (Liu and Rando, 2011). Similarly,
inducible blockade of the transcription factor NF-kB in aged
murine epidermis can abrogate cellular senescence and
restore the global gene expression program of old skin to
resemble that of young skin (Adler et al., 2007). An important
question is whether similar plasticity exists in human skin,
where aging occurs over decades rather than over months or
years as seen in model organisms. Defining clinically viable
strategies to unlock the plasticity of human aging is a critical
challenge.
An ideal technology to test this concept is broadband light
(BBL), also known as intense pulse light, a commonly available
and popular treatment to ‘‘rejuvenate’’ the skin. According to
the American Society for Aesthetic Plastic Surgery, over $215
million dollars were spent in the United States in 2009 on
these procedures. Unlike ablative light-based treatments that
improve the overall appearance of aged skin through thermal
destruction and regrowth of the epidermis and superficial
dermis, BBL uses a broad band of noncoherent light waves,
ranging from 560 to 1,200 nm, that are absorbed by a number
of components in the skin. Currently, BBL procedures are used
to decrease the appearance of fine rhytides, dyspigmentation,
erythema, and elastosis (Bitter Jr, 2000; Negishi et al., 2001).
Nevertheless, the molecular changes that are induced by this
treatment are unclear.
‘‘Rejuvenation’’ is a term that has been used by many
investigators and the lay public with different meanings, and thus
needs to be carefully defined. Here we define ‘‘rejuvenation’’ as
the restoration of characteristics of youthfulness to aged cells and
tissues. After BBL treatment, is the skin truly ‘‘rejuvenated’’ at a
molecular level, i.e., more closely resembles younger skin, or is
the treatment merely inducing a wounding or scarring response
that differs fundamentally from uninjured youthful skin?
ORIGINAL ARTICLE
394 Journal of Investigative Dermatology (2013), Volume 133 & 2013 The Society for Investigative Dermatology
Received 19 February 2012; revised 19 June 2012; accepted 11 July 2012;
published online 30 August 2012
This study was accepted as a poster presentation at the 2012 Society of
Investigative Dermatology Annual Meeting
1
Department of Dermatology, Stanford University School of Medicine,
Redwood City, California, USA;
2
Advanced Aesthetic Dermatology, Los
Gatos, California, USA and
3
Howard Hughes Medical Institute, Stanford,
California, USA
Correspondence: Anne Lynn S. Chang, Department of Dermatology, Stanford
University School of Medicine, 450 Broadway Street, MC 5334, Redwood
City, California 94063, USA. E-mail: alschang@stanford.edu
Abbreviations: BBL, broadband light; GO, Gene Ontology; lncRNA, long
noncoding RNA; polyA, polyadenylated; qRT–PCR, quantitative reverse
transcription–PCR; RG, rejuvenated gene; 3-seq, 3
0
-end sequencing for
expression quantification
Histologically, BBL has been reported to diminish melanin
deposition in the dermis and reduce telangiectasias (Bitter Jr,
2000; Prieto et al., 2002), with some reports also reporting an
increase in new upper papillary dermal collagen formation at
3 weeks after treatment (Negishi et al., 2001). However, this
neocollagen formation may be a more variable or short-term
effect, as ultrastructural analyses of skin 3 months after
treatment have not shown any collagen or elastin fiber effects
(Prieto et al., 2002). We examine the molecular basis of the
BBL treatment response by defining the global gene expres-
sion programs of photoaged and intrinsically aged human
skin and response to BBL. The intent is to capture the
broadest spectrum of changes in RNA induced by aging and
BBL, including alterations in gene expression (coding and
noncoding) and gene regulation.
RESULTS
Clinical and histologic changes after BBL treatment
To gain insights into the gene expression program associated
with skin aging and BBL treatment, we used skin biopsies
from young female volunteers (age o30 years, n ¼ 5) and
site-matched untreated and treated skin of aged female
volunteers (age 450 years, n ¼ 5), the latter after three
courses of monthly BBL treatment (n ¼ 5; Figure 1a). The
treated subjects were healthy older females with moderate to
severe photodamage on the forearms, and resided in the
Santa Clara or San Jose, California metropolitan area, where
on average there are 257 sunny days out of 365 days, with the
average UV Index being 5.1 (average UV Index in the United
States is 4.3; source: www.bestplaces.net, accessed 25 April
2012). Tanning beds, topical retinoids, or any other skin
treatments on the arms were prohibited for 1 month before
enrollment and during the study. During the study, the
participants were instructed to sun-protect their arms with a
broad-spectrum sunscreen and long-sleeved clothing, as well
as avoid prolonged sun exposure. The untreated young
subjects had the same inclusion criteria, but did not have
evidence of photoaging on the arms.
After three BBL treatments, arm skin showed improve-
ments in clinical ratings of intrinsic and extrinsic skin aging
parameters: fine wrinkling ( P ¼ 0.03), abnormal pigmentation
(P ¼ 0.02), and global skin aging assessment (P ¼ 0.01; Figure
1a–c). On histologic examination, the elastotic fibers in the
treated aged samples were found to be diminished and less
distinct compared with those in untreated aged samples
(Figure 1d–g). The periodic acid–Schiff stain showed no
obvious changes in collagen quantity in the dermis between
treated and untreated aged samples, although they did appear
less disordered after treatment (Figure 1h and i). The treated
aged samples also displayed subjective increases in epider-
mal thickness (Figure 1e, g and i) compared with untreated
aged samples (Figure 1d, f and h).
Expression program of coding and noncoding RNAs in aging
skin
Although gene expression programs of aging in several tissues
have been previously examined by microarray hybridization,
we used 3
0
-end sequencing for expression quantification
(3-seq), an efficient strategy of deep sequencing of RNA
3
0
ends (Tariq et al., 2011). The potential advantages of 3-seq
include accurate quantification of transcript levels not
obscured by cross-hybridization, an ability to determine
alterations in RNA termination and processing, and the ability
to discover previously unannotated genes, such as long
noncoding RNAs (lncRNAs). We generated 6.5–12.4 million
uniquely mappable reads for each sample, and identified
differentially expressed transcripts using DESeq algorithm
(see Materials and Methods).
To rigorously define aging in molecular terms, we first
identified transcript alterations associated with aging by com-
paring untreated young with untreated aged samples, and then
tested how BBL treatment to aged skin affected these parameters.
Comparison of mRNA transcript levels in untreated young
versus untreated aged, as well as untreated aged versus
treated aged, samples revealed a consistent significant
change in the expression level in 3,530 genes (Figure 2a).
The directionality of the gene expression change with BBL
treatment is shown in Figure 2a, with blue indicating a 2-fold
decrease and yellow indicating a 2-fold increase. Genes
whose transcript levels changed significantly between un-
treated young and untreated aged (n ¼ 2,265) are shown in
Supplementary Table S1 online.
To visually display the locations of significant genes on the
large heat map (Figure 2a), we have provided columns (in
magenta) to the right of the large heat map that represent
biological themes, according to Gene Ontology (GO) terms.
For instance, the ‘‘rejuvenated genes’’ (RGs) and lncRNAs
are distributed on both the upper and lower parts of the
large heat map. In contrast, the ‘‘immune response’’ genes
and ‘‘translation’’ genes are located on the lower half of
the heat map. The ‘‘cell adhesion’’ genes are located on
the upper half of the large heat map and are decreased
in the untreated young group, increased in the untreated
aged group, and intermediate in the treated aged group. The
magenta columns hence provide a general sense of what
biological function is altered and in what direction (increased
(yellow) or decreased (blue)), enabling comparison between
untreated aged, treated aged, and untreated young in the
large heat map. For instance, both the treated older samples
and the young untreated samples show increased transcript
levels in ‘‘immune response’’ and ‘‘translation,’’ as both these
groups are ‘‘up’’ (yellow). In contrast, the untreated aged
group shows decreased transcript levels, or ‘‘down’’ (blue) in
‘‘immune response’’ and ‘‘translation’’ genes compared with
the other two groups.
The gene programs associated with aging are multifaceted,
and are enriched for several biological themes. The top five
most significant GO terms that are increased in the aged
untreated compared with young untreated group included
translation (P ¼ 4.7 10
12
), translational elongation
(P ¼ 5.1 10
7
), macromolecular complex assembly (P ¼ 7.5
10
6
), ncRNA metabolic processing (P ¼ 6.2 10
6
),
and RNA processing (P ¼ 2.5 10
6
). The top five GO
terms that decreased in the aged untreated group
compared with the young untreated group were genes
encoding functions related to cell adhesion (P ¼ 1.5 10
17
),
www.jidonline.org 395
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL
biological adhesion (P ¼ 1.7 10
17
), homophilic cell adhe-
sion (P ¼ 7.8 10
8
), skeletal system development (P ¼ 3.2
10
7
), and enzyme-linked receptor protein signaling pathway
(P ¼ 5.2 10
6
). These gene sets are reminiscent of gene
expression changes associated with aging in other tissues and
organisms. For instance, translation-related genes or regula-
tion of translation affects aging in Caenorhabditis elegans
(Long et al., 2002) and Drosophila melanogaster (Kirby et al.,
2002). In addition, translation is believed to underlie
the important role of the TOR (target of rapamycin) pathway
in stem cell aging (Chen et al., 2009; Nelson et al., 2009;
Liu and Rando, 2011; Serrano, 2011).
BBL treatment promotes the gene expression pattern of young
skin
Genes whose average expression level in aged treated skin
was closer to young untreated skin than aged untreated skin
were defined as RGs. Specifically, mean gene expression
levels in the treated aged group were subtracted from mean
gene expression levels in the untreated young group as well
as from the untreated aged group. If the difference in gene
expression level was less with the untreated young group
compared with the difference with the untreated aged group,
the gene was operationally defined as ‘‘rejuvenated’’. A total
of 1,293 transcripts qualified as RGs (Supplementary Table S2
online). Hierarchical clustering showed that the gene
expression pattern of treated aged skin more closely
resembled that of untreated young skin than untreated aged
skin from the same individuals (Figure 2a). The RGs reflect
coherent biological themes and include genes that fall under
the following top six most significant GO terms: translation
(P ¼ 5.8 10
11
), RNA processing (P ¼ 6.3 10
8
), ncRNA
metabolic processing (P ¼ 1.4 10
7
), regulation of
cellular protein metabolic process (P ¼ 1.6 10
5
), cellular
Untreated
(n =5)
Mean score
(SD)
P
Clinical
parameter
Fine wrinkles
3.2 (1.3)
1.0 (1.0)
0.02
0.18
0.02
0.01
1.8 (2.5)
3.4 (2.3)
3.4 (1.5)
0 (0)
7.2 (1.3)
6.6 (1.1)
Treated
(n =5)
Mean score
(SD)
Coarse
wrinkles
Abnormal
pigmentation
Global
assessment
Figure 1. Clinical and histologic effects of broadband light (BBL) treatment. (a) Arm of a 73-year old female before BBL treatment (dashed box indicates area to
be treated and bandag e indicates untreated skin). (b) The same forearm after three BBL treatments with reduced fine wrinkling, hyperpigmentation, and erythema
in the treated area (dashed box) compared with the untreated area. (c) Skin aging parameters show significant decreases in fine wrinkling, abnormal
pigmentation, and global skin aging assessment after BBL treatment. The P-value by two-sided t-test. (d) Histology of skin before BBL treatment shows elastosis
(original magnification 200, hematoxylin and eosin (H&E) stain) and (e) reduced elastosis (original magnification 200, H&E stain) after BBL treatment.
(f) Before treatment, elastosis is prominent (original magnification 200, von Giesen stain). (g) After treatment, elastosis is less distinct (original magnification
200, von Giesen stain). (h) Before treatment, collagen fibers appear attenuated and disordered (original magnification 200, periodic acid–Schiff (PAS) stain).
(i) After treatment, collagen fibers are more uniform (original magnification 200, PAS stain). Bars ¼ 1 mm each.
396 Journal of Investigative Dermatology (2013), Volume 133
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL
macromolecular catabolic process (P ¼ 2.1 10
5
), and cell
cycle ( ¼ 2.4 10
5
; (Figure 2b, upper right).
A closer inspection of genes with expression patterns that
were ‘‘rejuvenated’’ by BBL treatment revealed several key
regulators known to control organismal aging (Figure 2c).
These include ZMPSTE24, a metalloproteinase that processes
lamin A, the gene defective in the dramatic premature aging
syndrome, Hutchinson-Guilford progeria. In addition, the
IGF1R receptor was one of the RGs identified, and this gene
product is directed linked to aging and longevity in human
beings, mice, and other model organisms (Liang et al., 2011;
Tazearslan et al., 2011), as well as in other model organisms.
Other RGs include EIF4G1 and EIF4EBP1, which are
associated with increased lifespan in C. elegans (Curran
and Ruvkun, 2007). MLL is a transcription regulator that
associates with telomeres (Caslini et al., 2009), and methy-
lates H3K4, which is required for normal lifespan in C.
elegans (Greer et al., 2010). MAP3K5 (ASK10) regulates
kinase activity in response to oxidative stress in a Klotho
aging mouse model (Hsieh et al., 2010). PSMD8 is a
proteasome component, and proteasome malfunction has
been reported to contribute to aging in human skin (Hwang
et al ., 2007). RING1 and MOV10 are in the Polycomb
pathway, which controls the lifespan of human fibroblasts
(Itahana et al., 2003). EEF2 (eukaryotic translation elongation
factor 2) is also an RG, and has been reported to associate
with age-related declines in protein synthesis in rats (Parado
et al., 1999). Finally, a number of tumor-suppressor genes
that are cell-cycle checkpoints and ensure genome integrity,
such as ING4 tumor suppressor, DAXX, and MSH2, are also
RGs. Thus, BBL treatment appears to be capable of restoring
many molecular features of youthful skin to aged human skin,
at least in the short term. Notably, we did not see gene
expression changes associated with wounding or scarring.
To confirm the findings on 3-seq, we performed quanti-
tative reverse transcription–PCR (qRT–PCR) to confirm the
“Rejuvenated”
IncRNAs
Immune response
Translation
Cell adhesion
1U
3U
2U
2T
5U
4U
3T
1T
4T
5T
1Y
3Y
4Y
2Y
5Y
1U
3U
2U
2T
5U
4U
3T
1T
4T
5T
1Y
3Y
4Y
2Y
5Y
Examples of “rejuvenated” genes
with known aging function
ZMPSTE24
IGF1R
ING4
EEF2
EIF4G1
EIF3B
MLL
EIF4EBP1
RING1
PSMD8
MOV10
MAP3K5
“Rejuvenated” genes’ top 6 most
significant GO terms
Translation
RNA processing
lncRNA metabolic processing
Regulation of cellular protein
Cellular macromolecule catabolic
Cell cycle
–Log (P-value)
–2 +2-Fold
Significant genes (n =3,530) among those that change with age and with treatment
Each gene (row) in large heat map corresponds to same gene (row)
in adjacent box on right, with magenta marks indicating distributions of genes
with biologic themes.
All samples
All samples
All samples
All samples
All samples
024681012
Figure 2. Effects of broadband light (BBL) treatment on coding and noncoding RNAs in aging skin. (a) Gene expression clustering of treated aged samples is
intermediate between untreated young and untreated aged samples. Transcript levels that significantly change with untreated young versus untreated aged
samples, as well as untreated aged versus treated aged samples (n ¼ 3,530 total transcripts), are shown. Columns indicate single subject sample and rows
indicate gene. T, aged treated; U, aged untreated; Y, young untreated. Magenta columns are visual representations of the gene distributions on the large heat
map (left) as grouped by biological function. For instance, ‘‘immune response’’ and ‘‘translation’’ related genes are on the lower half of the heat map, with
yellow indicating increased levels (or ‘‘up’’) in treated aged and untreated young groups; the corresponding location in the large heat map for immune response
and translation are blue (or ‘‘down’’) in the untreated aged group. Distributions on the large heat map of rejuvenated genes (RGs; n ¼ 1,293) and ‘‘long
noncoding RNAs’’ (lncRNAs) are shown in the first and second magenta columns, respectively. (b) The top six most significant Gene Ontology (GO) terms
among RGs. (c) Examples of RGs with known aging function.
www.jidonline.org 397
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL
levels of ZMPSTE24 on an independent group of untreated
women across a spectrum of ages. The 3-seq had showed that
untreated aged skin had the highest levels of ZMPSTE24
transcript expression level, treated aged skin had intermedi-
ate levels of ZMPSTE24 transcript level, and untreated young
skin had the lowest levels (Figure 3a). By qRT–PCR, untreated
aged arm skin (age 75 years) had the highest ZMPSTE24
transcript levels, untreated middle-aged arm skin (age 35
years) had intermediate levels, and untreated young arm skin
(age 24 years) had the lowest levels (Figure 3b). This gradient
has not been reported earlier in humans, but is an
independent indicator suggesting that our findings are of
biological relevance to physiological aging.
The enrichment in mRNAs encoding genes involved in
RNA processing prompted us to evaluate the expression
levels of additional RNA classes. The lncRNAs are a newly
recognized class of genetic elements that are pervasively
transcribed in the human genome (Wang et al., 2009;
Wapinski and Chang, 2011). The roles of lncRNAs in aging
and in skin have not been studied, as they have not been
represented on microarray platforms in the past. However,
the 3-seq technology can readily capture and quantify
lncRNA expression. Of the 3,530 transcripts with altered
levels between untreated young and untreated aged, 151 are
lncRNAs. The chromosomal locations and most proximate
genes of these lncRNAs are shown in Supplementary Table
S3 online. Of the 1,293 RGs, 42 were lncRNAs. The
chromosomal locations and most proximate genes of these
lncRNAs are listed in Supplementary Table S4 online, with
heat map in Supplementary Figure S1 online. These findings
suggest that lncRNAs are potentially involved in the process
of aging and rejuvenation, paralleling their roles in develop-
ment and cellular reprogramming (Gupta et al., 2010; Loewer
et al., 2010). Our data provide an initial set of lncRNAs
associated with human aging that sets the groundwork for
functional studies in the future.
GO analysis of the 151 lncRNAs with significant
difference in expression between young untreated and aged
untreated skin showed no significant enrichment for terms.
Similarly, GO analysis of the 42 ‘‘rejuvenated’’ lncRNAs
showed no significant enrichment for terms. However, the
importance of lncRNAs in ‘‘rejuvenation’’ is not necessarily
diminished. For instance, our 42 lncRNAs are a small number
and future studies with greater sample size may identify more
lncRNAs, enabling identification of significant GO terms. In
addition, lncRNAs are a new class of RNA, and our GO
analysis relied on the proximity of lncRNA sequences to
known genes; it is possible that lncRNAs are important for
regulating genes that are not necessarily proximal to the
lncRNA (Gupta et al., 2010).
The effect of BBL treatment on the immune response
includes altering the immune profile in a way that resembles
untreated young skin. Figure 2a shows that although genes
related to immune response are ‘‘up’’ after treatment, this
‘‘up’’ profile more closely resembles untreated young samples.
This suggests that at least a portion of the immune response
that is ‘‘up’’ after treatment is part of the ‘‘rejuvenated’’ profile
and not specific to being treated with BBL.
The NF-kB pathway had been shown to be important in
skin aging and rejuvenation (Adler et al., 2007), and we
found that the RGs are indeed highly enriched for genes
bound by NF-kB as measured by chromatin immunoprecipi-
tation sequencing experiments. In all, 827 of the 1,293 RGs
are bound by NF-kB(P ¼ 1.2 10
75
, hypergeometric test).
Interestingly, NF-kB itself was not one of the identified RGs.
RNA 3
0
termination appears unaffected by aging or BBL
The 3-seq captures the 3
0
polyadenylated (polyA) RNA
fragments for deep sequencing, and thus has the potential
to detect alterations in the location of 3
0
transcript termina-
tion. The 3-seq method samples RNA sequences immediately
upstream of the polyA tails. If there were changes in the use
of the polyA site within the last exon such that the last exon is
lengthened or truncated, this would be detected in the
sequencing reads. This method does not evaluate the length
of the polyA tail.
0
0.005
0.01
0.015
0.02
Relative ZMPSTE24
expression
Age (years):
3′ RNA sequencing
150 -
0
150 -
0
150 -
0
150 -
0
150 -
0
150 -
0
chr1: 40,500,000
40,530,00040,520,000
10 kb
a
b
ZMPSTE24
OldOldYoungYoung Old+BBLOld+BBL
40,510,000
Young
(24)
Middle aged
(35)
Old
(75)
Figure 3. ZMPSTE24 transcript levels increase after broadband light (BBL)
treatment. (a) Schematic of ZMPSTE24 locus on chromosome 1, hg18. The
3
0
-seq (deep sequencing of RNA 3
0
end) reads were plotted for two old
individuals with and without BBL treatment and two untreated young
samples. (b) ZMPSTE24 transcript expression is lower in untreated old skin
compared with untreated young skin by quantitative reverse transcription–
PCR (RT–qPCR). ZMPSTE24 transcript expression in untreated middle-aged
skin is intermediate (n ¼ 1).
398 Journal of Investigative Dermatology (2013), Volume 133
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL
Alternative 3
0
-end usage is an important regulatory
mechanism (Mayr and Bartel, 2009), and can alter gene
expression output by changing the content of the 3
0
untranslated region, which may then alter the repertoire of
microRNA targets or RNA-binding proteins (such as those
known to occur in cancer; Shapiro et al., 2011). Thus, in
addition to quantifying changes in transcript abundance, we
also searched for changes in transcript termination in
association with aging or BBL treatment. Systematic compar-
ison of all 3-seq reads showed that, as anticipated, the
majority of reads fell into the annotated last exon, i.e.,
o1,000 bp from the transcriptional stop site (Figure 3a), and
there were no consistent changes of 3
0
-end usage associated
with aging or BBL treatment (Figure 3b). For instance, if the
distribution of distances from transcriptional stop site for the
RNAs from young untreated and aged treated samples were
different, then aging may be associated with systemic
changes in mRNA 3
0
terminations (Figure 4).
Treatment-specific effects of BBL
In addition to affecting the age-associated gene expression
program, we also considered the possibility that BBL
treatment may induce unique treatment-specific effects that
are distinct from aging. For instance, BBL treatment could
induce wound healing or scarring response in addition to
rejuvenation effects. We identified consistent changes in the
expression of 1,112 genes that occur only in BBL-treated
samples but not in either untreated young or untreated aged
samples. Among these treatment-specific genes, the top five
GO term categories significantly associated with increased
expression after treatment were as follows: immune response
(P ¼ 3.8 10
12
), positive regulation of immune system
process (P ¼ 2.0 10
8
), cell activation (P ¼ 5.7 10
8
), T-
cell activation (6.0 10
7
), and defense response
(P ¼ 1.4 10
7
). These categories are suggestive of an
immune response to BBL separate from the immune response
genes that are also increased in untreated young samples (as
mentioned in the above section). The top five GO term
categories significantly associated with decreased expression
after treatment were as follows: regulation of transcription
(P ¼ 2.0 10
6
), transcription (P ¼ 1.7 10
5
), response to
organic substance (P ¼ 1.1 10
4
), response to hormone
stimulus (P ¼ 4.4 10
4
), and negative regulation of tran-
scription (P ¼ 4.7 10
4
). These genes are distinct from a
previously described ‘‘wound signature’’ that characterizes
response to skin wounding (Chang et al., 2005); however, it is
difficult to directly compare signatures with those in our
study, as there are no published data at the equivalent time
point after wounding as used in this study (4 weeks).
Finally, the top 10 genes that are most highly upregulated
and downregulated in the treated aged samples compared
with the untreated aged samples are listed in Table 1.
DISCUSSION
Our results suggest that regulators of organismal aging can be
altered in human skin using commonly available BBL
technology. How such plasticity in aging may be modulated
for healthful benefits such as prevention or treatment of age-
associated skin conditions remains to be seen. Although BBL
technology has been harnessed for its ability to produce a
more clinically ‘‘youthful’’ appearance, our study suggests
that ‘‘rejuvenation’’ at a molecular level has also occurred,
with a number of genes linked to the aging process being
altered in expression after treatment to more closely resemble
young skin. Hence, it is possible that the clinical phenotype
represents a functional rejuvenation (at least in the short
term), rather than just a cosmetic mimic of youthful
appearance.
As the BBL technology has been in existence for o20
years, the long-term effects of BBL remain to be determined.
Although this study assessed the skin 4 weeks after treatment,
it is unclear how durable the clinical and molecular response
is. Also unknown is whether there is a decrease of age-
associated skin changes such as seborrheic keratosis or
actinic keratosis with time. It may be informative to follow
these current participants in the long term (e.g., 45 years)
with photographs and skin biopsies to determine the duration
of clinical, histologic, and molecular effect of BBL treatment.
The precise mechanisms by which BBL (noncoherent
wavelengths of light) alters gene expression are currently not
well understood. For instance, it is known that BBL is
absorbed by different targets including melanin and hemo-
globin, leading to decreased erythema and pigmentation. It is
thought that the decrease in fine wrinkling is partly due to the
production of new collagen (Fisher et al., 2008). However,
the genes identified in this study were not collagen specific. It
may be possible that if the posttreatment skin biopsies were per-
formed earlier than 4 weeks, some of the gene expression changes
related to collagen production might have been captured.
Gene expression programs associated with human aging
appear to differ between organ types. For instance, the aging
human kidney and human muscle seem to have distinct
gene expression signatures (Rodwell et al., 2004; Zahn
et al., 2006). The aging gene expression profiles of human
skin generated in this study do not appear to be the same as
Table 1. Top 10 most significantly changed gene
expression levels overall between BBL-treated aged
samples and untreated aged samples
Gene symbol
Fold change: treated aged
versus untreated aged
Directionality
of change
HEPHL1 3.19 Down
ZNF660 3.09 Down
LY6G6D 2.53 Down
COCH 2.38 Down
CCL18 2.36 Up
CEP78 2.36 Down
ANGPTL7 2.34 Down
SLN 2.17 Down
CPXM1 2.10 Up
SAMD5 2.08 Down
Abbreviation: BBL, broadband light.
www.jidonline.org 399
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL
other reported organ types; however, future direct compar-
ison studies may shed more light on this issue.
NF-kB is an important regulator of gene expression in
many contexts. In this case, the most relevant role of NF-kBis
likely in controlling cell senescence (Bernard et al., 2004;
Adler et al., 2007) and immune response. Our finding that
RGs are highly enriched for NF-kB-bound genes suggests that
BBL may influence pathways controlled by NF-kB. The
precise mechanisms by which this occurs remain to be
investigated. Nevertheless, our results are consistent with a
prior study showing that inducible blockade of NF-kB in aged
murine skin restores the gene expression program and
phenotypes of young skin (Adler et al., 2007).
It is difficult to directly compare the results of our study
with the gene expression profiles in humans reported
currently in the literature for two reasons: (1) the time point
of biopsies may not be exactly the same, and (2) the nature of
the disease entity or treatment is not the same as BBL. For
instance, gene expression patterns in human postburn
hypertrophic scars at 6–15 months in two pediatric and two
adult patients identified six genes as significantly increased
(Paddock et al., 2003), none of which were significantly
changed in our BBL study. In another example, an in vitro
human keratinocyte model using scratch wounding has
shown increased activation of NF-kB in cells between
1 and 14 days (Adams et al., 2007). Our study captured the
1-month time point when the effects of wound healing might
be decreasing, and we are more likely to detect rejuvenation
effects. At our 1-month time point, NF-kB levels were not
significantly increased, but genes known to interact with
NF-kB were significantly increased, which is a distinct and,
to our knowledge, previously unreported finding.
Two RGs, RING1 and MOV10, are in the Polycomb
pathway, with the potential to contribute to both rejuvenation
effects and wound repair. In mice and cell culture, the
Polycomb pathway controls the lifespan of human fibroblasts
(Itahana et al., 2003) and associates with the upregulation of
wound repair genes (Shaw and Martin, 2009).
The ligands for Toll-like receptors 2, 3, and 5 have been
reported to affect the transcript and protein levels of matrix
metalloproteinases 1 and 9 and induce the nuclear transloca-
tion of NF-kB after 24–48 hours in human keratinocyte
culture (Lee et al., 2009). We did not detect significant
increases in Toll-like receptors 2, 3, and 5, or NF-kB, but
our study was in vivo and skin samples were obtained at the
1-month time point.
In addition, although our data show that coherent
biological themes such as ‘‘translation’’ or ‘‘RNA processing’’
are altered after BBL treatment, our study does not identify the
population of cells within the skin that undergo these changes.
Future studies that get at this question may better explain how
BBL treatment might lead to histological or structural changes
such as resorption of elastosis or collagen deposition.
Figure 4. Broadband light (BBL) treatment and aging show no systematic
changes of 3
0
-end usage. (a) Systematic comparison of all 3-seq (deep
sequencing of RNA 3
0
end) reads showing that the majority of reads fell into
the annotated last exon (based on distance of within 1,000 bp from
transcriptional start site (TSS)) for untreated aged, treated aged, and untreated
young groups. The y-axis shows the average intensity of the 3-seq signal.
(b) There were no systematic changes of 3
0
-end usage associated with aging or
BBL treatment, as the reads showed similar length distributions between the
untreated aged, treated aged, and untreated young groups.
Aged untreated
+1
Distance from transcriptional stop site (kb)
Average diagram
Average intensity
Distance from TSS (bp)
2.0
1.5
1.0
0.5
0.0
Aged untreated
Aged treated
Young
–1,500
–500 0
1,500
1,000500
–1,000
–1
0
YoungAged treated
400 Journal of Investigative Dermatology (2013), Volume 133
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL
It would be interesting to compare whether other
modalities known to reduce clinical skin aging parameters
such as topical tretinoin result in gene expression changes
that are in common with BBL-induced changes.
In addition, comparison of non-sun-exposed older skin
before and after treatment may identify gene expression
changes that are specific to intrinsic skin aging.
This is an exploratory study, and we will consider
including treated young skin in future studies. In this study,
we did not treat younger skin (defined as age o30 years
for this study) because there was no clinical indication;
these subjects did not have detectable photoaging or intrinsic
aging on the arm skin. As it is unlikely that BBL would be used
in practice on young skin without photoaging (except possibly
for hair removal), we did not include this group in the study.
The current literature on the ability of BBL to induce
collagen neogenesis is contradictory. Although some reports
on histologic changes induced by BBL include collagen
neogenesis (Negishi et al., 2001), there are other studies
showing no change (Prieto et al., 2002). This latter study also
reported no change in elastin content after treatment. In our
study, there were no marked changes in collagen content
after treatment on periodic acid–Schiff staining. There were
decreases in the amount of elastin on von Giesen staining.
We did not detect any significant changes in collagen or
elastin gene expression levels after treatment. One possibility
is that the histology was taken at a single time point, and may
not have captured the time when collagen or elastin
expression levels were more markedly changed. Future
studies will indeed biopsy-treated skin longitudinally to
reveal the kinetics of activation/suppression of target genes.
In addition, it is precisely the goal of this study to extend
beyond the conventional histologic analysis of skin and
explore molecular changes of skin aging and BBL treatment.
We observed numerous gene expression changes related to
pathways beyond connective tissue organization that can be
modulated by BBL.
Finally, future studies with larger sample size may enable
us to identify additional significant genes (both coding and
noncoding) whose expression is altered in untreated young
versus untreated aged, as well as untreated aged and treated
aged human skin samples. Larger sample size might also
enable us to correlate the degree of clinical response with
more ‘‘rejuvenated’’ gene expression changes.
MATERIALS AND METHODS
Human subjects and sample acquisition
This study was conducted in accordance with the Declaration of
Helsinki Principles. After Institutional Review Board approval and
written informed consent was obtained, five female participants over
the age of 50 years underwent BBL treatments to the left forearm.
Inclusion criteria included Fitzpatrick skin type II or III, and a global
assessment of forearm skin aging consistent with moderate or severe
forearm skin aging (modified validated instrument from McKenzie
et al., 2010) for treated participants. Treatments were performed on
the Sciton Joule Platform using the BBL module. The same investigator
performed the treatments at 4-week intervals for a total of three
treatments using a 515-nm or a 560-nm cutoff filter at a single long
pulse of 10–20ms duration, with fluences of 8–14 J cm
2
.Ateach
treatment session, two or more passes were performed. At 4 weeks
after the third BBL treatment, 4-mm skin biopsies were performed by
the Keys punch technique from the treated and adjacent untreated
skin. Punch biopsies (4 mm) were taken from non-sun-exposed arm
skin of five participants o30 years old. These specimens were
bisected and placed into either RNAlater (Ambion, cat. no. AM7022,
Grand Island, NY) or formalin solution for staining with hematoxylin
and eosin, von Giesen, or periodic acid–Schiff.
The 3-seq and bioinformatics
Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit
(Qiagen, Germantown, MD). The 3-seq was performed as described in
Beck et al. (2010). In brief, oligo-dT-directed reverse transcription
generated complementary DNAs corresponding to 3
0
ends of polyA
transcripts; the complementary DNAs were cloned and subjected to
deep sequencing on the Illumina GAIIx (San Diego, CA) platform with
raw read length of 36 bp. Raw reads were aligned to human genome
(hg18) using bowtie (Langmead et al., 2009); each sample generated
6.5–12.4 million uniquely mappable reads. The 3
0
sequencing of skin
transcripts was performed to assess length distributions.
Reads per kilobase of exon per million mappable reads (RPKM, a
direct measure of transcript abundance) and the number of raw reads
falling on to each gene were calculated using a self-developed script
by Kun Qu. The Reference Sequence (RefSeq; www.ncbi.nlm.nih.-
gov/RefSeq) and Ensembl (http://www.ensembl.org) annotated non-
coding genes were included. Significant genes were called using the
DESeq package (http://www.bioconductor.org) comparing aged
treated with aged untreated samples (genes changed because of
treatment), and aged untreated with young untreated samples (genes
changed because of aging). Unsupervised hierarchical clustering of
significantly different expressed genes was performed using Cluster.
The GO terms were generated using DAVID (Database for Annotation,
Visualization and Integrated Discovery) Bioinformatics Resources 6.7
(http://david.abcc.ncifcrf.gov/). Genes close to lncGenes were identified
using GREAT database (http://great.stanford.edu). These data have
been deposited into the Gene Expression Omnibus.
To determine the overlap between RGs and NF-kB binding, we
downloaded the NF-kB-bound genes identified by the ENCODE
project (ENCODE Consortium, 2011) by chromatin immunopreci-
pitation sequencing experiments. A total of 9,650 genes bound NF-
kB in one or more cell types, and these were compared with the RG
gene list.
RT–qPCR
Total RNA was extracted with TRIzol (Invitrogen, Grand Island, NY)
followed by RNeasy column purification (Qiagen) and DNAse
Turbo Treatment (Ambion). RT–qPCR was performed using total
RNA (10 ng), Taqman One Step RT–PCR master mix, and one
of the following Taqman assays: GAPDH (Hs99999905_m1) and
ZMPSTE24 (Hs00956778_m1; Applied Biosystems, Carlsbad, CA).
Reactions were in triplicate for each sample and were performed a
minimum of two times. Data were normalized to glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) levels.
CONFLICT OF INTEREST
PB has given lectures on broadband light technology. The other authors state
no conflict of interest.
www.jidonline.org 401
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL
ACKNOWLEDGMENTS
This study was funded by a research grant from Sciton. We are indebted to
Paul Khavari and Jean Tang for prereview of the manuscript. We thank Olena
Mykhaylichenko and Sarah Jacobs for administrative support.
SUPPLEMENTARY MATERIAL
Supplementary material is linked to the online version of the paper at http://
www.nature.com/jid
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Rejuvenation of Gene Expression in Aging Skin by BBL