Rejuvenation of gene expression pattern of aged human skin by broadband light treatment: a pilot study

Rejuvenation of Gene Expression Pattern ofAged Human Skin by Broadband Light Treatment:A Pilot StudyAnne Lynn S. Chang1, Patrick H. Bitter Jr2, Kun Qu1, Meihong Lin1, Nicole A. Rapicavoli1,3 and Howard Y. Chang1,3 Studies in model organisms suggest that aged cells can be functionally rejuvenated, but whether this conceptapplies to human skin is unclear. Here we apply 30-end sequencing for expression quantification (‘‘3-seq'') todiscover the gene expression program associated with human photoaging and intrinsic skin aging (collectivelytermed ‘‘skin aging''), and the impact of broadband light (BBL) treatment. We find that skin aging was associatedwith a significantly altered expression 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 proximallong noncoding RNAs. Skin aging is not associated with systematic changes in 30-end mRNA processing. Hence,BBL treatment can restore gene expression pattern of photoaged and intrinsically aged human skin to resembleyoung skin. In addition, our data reveal, to our knowledge, a previously unreported set of targets that may leadto 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 murine epidermis can abrogate cellular senescence and Aging is under complex genetic and environmental control.
restore the global gene expression program of old skin to Aging is associated with large-scale changes in gene resemble that of young skin (Adler et al., 2007). An important expression, and how such changes may be modulated for question is whether similar plasticity exists in human skin, healthful benefits in human beings is not clear. Numerous where aging occurs over decades rather than over months or single-gene mutations have been identified that can extend years as seen in model organisms. Defining clinically viable the lifespan of model organisms (Partridge, 2010; de strategies to unlock the plasticity of human aging is a critical Magalhaes et al., 2012), and dietary restriction can slow the rate of aging, even if applied late in life (Partridge, 2010).
An ideal technology to test this concept is broadband light More recently, several interventions have been shown to (BBL), also known as intense pulse light, a commonly available confer features of youthfulness to aged cells or tissues, and popular treatment to ‘‘rejuvenate'' the skin. According to demonstrating a remarkable plasticity of the aging process.
the American Society for Aesthetic Plastic Surgery, over $215 For instance, heterochronic parabiosis between young and million dollars were spent in the United States in 2009 on old mice enables circulatory factors to restore the functions of these procedures. Unlike ablative light-based treatments that aged muscle stem cells (Liu and Rando, 2011). Similarly, improve the overall appearance of aged skin through thermal inducible blockade of the transcription factor NF-kB in aged destruction and regrowth of the epidermis and superficialdermis, BBL uses a broad band of noncoherent light waves,ranging from 560 to 1,200 nm, that are absorbed by a number 1Department of Dermatology, Stanford University School of Medicine, of components in the skin. Currently, BBL procedures are used Redwood City, California, USA; 2Advanced Aesthetic Dermatology, Los to decrease the appearance of fine rhytides, dyspigmentation, Gatos, California, USA and 3Howard Hughes Medical Institute, Stanford, erythema, and elastosis (Bitter Jr, 2000; Negishi et al., 2001).
Nevertheless, the molecular changes that are induced by this This study was accepted as a poster presentation at the 2012 Society of treatment are unclear.
Investigative Dermatology Annual Meeting ‘‘Rejuvenation'' is a term that has been used by many Correspondence: Anne Lynn S. Chang, Department of Dermatology, StanfordUniversity School of Medicine, 450 Broadway Street, MC 5334, Redwood investigators and the lay public with different meanings, and thus City, California 94063, USA. E-mail: [email protected] needs to be carefully defined. Here we define ‘‘rejuvenation'' as Abbreviations: BBL, broadband light; GO, Gene Ontology; lncRNA, long the restoration of characteristics of youthfulness to aged cells and noncoding RNA; polyA, polyadenylated; qRT–PCR, quantitative reverse tissues. After BBL treatment, is the skin truly ‘‘rejuvenated'' at a transcription–PCR; RG, rejuvenated gene; 3-seq, 30-end sequencing for molecular level, i.e., more closely resembles younger skin, or is the treatment merely inducing a wounding or scarring response Received 19 February 2012; revised 19 June 2012; accepted 11 July 2012;published online 30 August 2012 that differs fundamentally from uninjured youthful skin? Journal of Investigative Dermatology (2013), Volume 133 & 2013 The Society for Investigative Dermatology ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL Histologically, BBL has been reported to diminish melanin (3-seq), an efficient strategy of deep sequencing of RNA deposition in the dermis and reduce telangiectasias (Bitter Jr, 30 ends (Tariq et al., 2011). The potential advantages of 3-seq 2000; Prieto et al., 2002), with some reports also reporting an include accurate quantification of transcript levels not increase in new upper papillary dermal collagen formation at obscured by cross-hybridization, an ability to determine 3 weeks after treatment (Negishi et al., 2001). However, this alterations in RNA termination and processing, and the ability neocollagen formation may be a more variable or short-term to discover previously unannotated genes, such as long effect, as ultrastructural analyses of skin 3 months after noncoding RNAs (lncRNAs). We generated 6.5–12.4 million treatment have not shown any collagen or elastin fiber effects uniquely mappable reads for each sample, and identified (Prieto et al., 2002). We examine the molecular basis of the differentially expressed transcripts using DESeq algorithm BBL treatment response by defining the global gene expres- (see Materials and Methods).
sion programs of photoaged and intrinsically aged human To rigorously define aging in molecular terms, we first skin and response to BBL. The intent is to capture the identified transcript alterations associated with aging by com- broadest spectrum of changes in RNA induced by aging and paring untreated young with untreated aged samples, and then BBL, including alterations in gene expression (coding and tested how BBL treatment to aged skin affected these parameters.
noncoding) and gene regulation.
Comparison of mRNA transcript levels in untreated young versus untreated aged, as well as untreated aged versus treated aged, samples revealed a consistent significant Clinical and histologic changes after BBL treatment change in the expression level in 3,530 genes (Figure 2a).
To gain insights into the gene expression program associated The directionality of the gene expression change with BBL with skin aging and BBL treatment, we used skin biopsies treatment is shown in Figure 2a, with blue indicating a 2-fold from young female volunteers (age o30 years, n ¼ 5) and decrease and yellow indicating a 2-fold increase. Genes site-matched untreated and treated skin of aged female whose transcript levels changed significantly between un- volunteers (age 450 years, n ¼ 5), the latter after three treated young and untreated aged (n ¼ 2,265) are shown in courses of monthly BBL treatment (n ¼ 5; Figure 1a). The Supplementary Table S1 online.
treated subjects were healthy older females with moderate to To visually display the locations of significant genes on the severe photodamage on the forearms, and resided in the large heat map (Figure 2a), we have provided columns (in Santa Clara or San Jose, California metropolitan area, where magenta) to the right of the large heat map that represent on average there are 257 sunny days out of 365 days, with the biological themes, according to Gene Ontology (GO) terms.
average UV Index being 5.1 (average UV Index in the United For instance, the ‘‘rejuvenated genes'' (RGs) and lncRNAs States is 4.3; source: www.bestplaces.net, accessed 25 April are distributed on both the upper and lower parts of the 2012). Tanning beds, topical retinoids, or any other skin large heat map. In contrast, the ‘‘immune response'' genes treatments on the arms were prohibited for 1 month before and ‘‘translation'' genes are located on the lower half of enrollment and during the study. During the study, the the heat map. The ‘‘cell adhesion'' genes are located on participants were instructed to sun-protect their arms with a the upper half of the large heat map and are decreased broad-spectrum sunscreen and long-sleeved clothing, as well in the untreated young group, increased in the untreated as avoid prolonged sun exposure. The untreated young aged group, and intermediate in the treated aged group. The subjects had the same inclusion criteria, but did not have magenta columns hence provide a general sense of what evidence of photoaging on the arms.
biological function is altered and in what direction (increased After three BBL treatments, arm skin showed improve- (yellow) or decreased (blue)), enabling comparison between ments in clinical ratings of intrinsic and extrinsic skin aging untreated aged, treated aged, and untreated young in the parameters: fine wrinkling (P ¼ 0.03), abnormal pigmentation large heat map. For instance, both the treated older samples (P ¼ 0.02), and global skin aging assessment (P ¼ 0.01; Figure and the young untreated samples show increased transcript 1a–c). On histologic examination, the elastotic fibers in the levels in ‘‘immune response'' and ‘‘translation,'' as both these treated aged samples were found to be diminished and less groups are ‘‘up'' (yellow). In contrast, the untreated aged distinct compared with those in untreated aged samples group shows decreased transcript levels, or ‘‘down'' (blue) in (Figure 1d–g). The periodic acid–Schiff stain showed no ‘‘immune response'' and ‘‘translation'' genes compared with obvious changes in collagen quantity in the dermis between the other two groups.
treated and untreated aged samples, although they did appear The gene programs associated with aging are multifaceted, less disordered after treatment (Figure 1h and i). The treated and are enriched for several biological themes. The top five aged samples also displayed subjective increases in epider- most significant GO terms that are increased in the aged mal thickness (Figure 1e, g and i) compared with untreated untreated compared with young untreated group included aged samples (Figure 1d, f and h).
(P ¼ 4.7  1012), (P ¼ 5.1  107), macromolecular complex assembly (P ¼ 7.5 Expression program of coding and noncoding RNAs in aging  106), ncRNA metabolic processing (P ¼ 6.2  106), and RNA processing (P ¼ 2.5  106). The top five GO Although gene expression programs of aging in several tissues have been previously examined by microarray hybridization, compared with the young untreated group were genes we used 30-end sequencing for expression quantification encoding functions related to cell adhesion (P ¼ 1.5  1017),

ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL 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 tobe treated and bandage indicates untreated skin). (b) The same forearm after three BBL treatments with reduced fine wrinkling, hyperpigmentation, and erythemain the treated area (dashed box) compared with the untreated area. (c) Skin aging parameters show significant decreases in fine wrinkling, abnormalpigmentation, 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.
biological adhesion (P ¼ 1.7  1017), homophilic cell adhe- were defined as RGs. Specifically, mean gene expression sion (P ¼ 7.8  108), skeletal system development (P ¼ 3.2  levels in the treated aged group were subtracted from mean 107), and enzyme-linked receptor protein signaling pathway gene expression levels in the untreated young group as well (P ¼ 5.2  106). These gene sets are reminiscent of gene as from the untreated aged group. If the difference in gene expression changes associated with aging in other tissues and expression level was less with the untreated young group organisms. For instance, translation-related genes or regula- compared with the difference with the untreated aged group, tion of translation affects aging in Caenorhabditis elegans the gene was operationally defined as ‘‘rejuvenated''. A total (Long et al., 2002) and Drosophila melanogaster (Kirby et al., of 1,293 transcripts qualified as RGs (Supplementary Table S2 2002). In addition, translation is believed to underlie online). Hierarchical clustering showed that the gene the important role of the TOR (target of rapamycin) pathway expression pattern of treated aged skin more closely in stem cell aging (Chen et al., 2009; Nelson et al., 2009; resembled that of untreated young skin than untreated aged Liu and Rando, 2011; Serrano, 2011).
skin from the same individuals (Figure 2a). The RGs reflectcoherent biological themes and include genes that fall under BBL treatment promotes the gene expression pattern of young the following top six most significant GO terms: translation (P ¼ 5.8  1011), RNA processing (P ¼ 6.3  108), ncRNA Genes whose average expression level in aged treated skin (P ¼ 1.4  107), was closer to young untreated skin than aged untreated skin cellular protein metabolic process (P ¼ 1.6  105), cellular Journal of Investigative Dermatology (2013), Volume 133

ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL 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 "Rejuvenated" genes' top 6 most with biologic themes. significant GO terms lncRNA metabolic processing Regulation of cellular protein Cellular macromolecule catabolic –Log (P-value) Examples of "rejuvenated" genes with known aging function Figure 2. Effects of broadband light (BBL) treatment on coding and noncoding RNAs in aging skin. (a) Gene expression clustering of treated aged samples isintermediate between untreated young and untreated aged samples. Transcript levels that significantly change with untreated young versus untreated agedsamples, as well as untreated aged versus treated aged samples (n ¼ 3,530 total transcripts), are shown. Columns indicate single subject sample and rowsindicate gene. T, aged treated; U, aged untreated; Y, young untreated. Magenta columns are visual representations of the gene distributions on the large heatmap (left) as grouped by biological function. For instance, ‘‘immune response'' and ‘‘translation'' related genes are on the lower half of the heat map, withyellow indicating increased levels (or ‘‘up'') in treated aged and untreated young groups; the corresponding location in the large heat map for immune responseand 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 ‘‘longnoncoding RNAs'' (lncRNAs) are shown in the first and second magenta columns, respectively. (b) The top six most significant Gene Ontology (GO) termsamong RGs. (c) Examples of RGs with known aging function.
macromolecular catabolic process (P ¼ 2.1  105), and cell kinase activity in response to oxidative stress in a Klotho cycle ( ¼ 2.4  105; (Figure 2b, upper right).
aging mouse model (Hsieh et al., 2010). PSMD8 is a A closer inspection of genes with expression patterns that proteasome component, and proteasome malfunction has were ‘‘rejuvenated'' by BBL treatment revealed several key been reported to contribute to aging in human skin (Hwang regulators known to control organismal aging (Figure 2c).
et al., 2007). RING1 and MOV10 are in the Polycomb These include ZMPSTE24, a metalloproteinase that processes pathway, which controls the lifespan of human fibroblasts lamin A, the gene defective in the dramatic premature aging (Itahana et al., 2003). EEF2 (eukaryotic translation elongation syndrome, Hutchinson-Guilford progeria. In addition, the factor 2) is also an RG, and has been reported to associate IGF1R receptor was one of the RGs identified, and this gene with age-related declines in protein synthesis in rats (Parado product is directed linked to aging and longevity in human et al., 1999). Finally, a number of tumor-suppressor genes beings, mice, and other model organisms (Liang et al., 2011; that are cell-cycle checkpoints and ensure genome integrity, Tazearslan et al., 2011), as well as in other model organisms.
such as ING4 tumor suppressor, DAXX, and MSH2, are also Other RGs include EIF4G1 and EIF4EBP1, which are RGs. Thus, BBL treatment appears to be capable of restoring associated with increased lifespan in C. elegans (Curran many molecular features of youthful skin to aged human skin, and Ruvkun, 2007). MLL is a transcription regulator that at least in the short term. Notably, we did not see gene associates with telomeres (Caslini et al., 2009), and methy- expression changes associated with wounding or scarring.
lates H3K4, which is required for normal lifespan in C.
To confirm the findings on 3-seq, we performed quanti- elegans (Greer et al., 2010). MAP3K5 (ASK10) regulates tative reverse transcription–PCR (qRT–PCR) to confirm the ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL levels of ZMPSTE24 on an independent group of untreated 3′ RNA sequencing 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 35years) 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 anindependent 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 newlyrecognized 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 alteredlevels between untreated young and untreated aged, 151 are lncRNAs. The chromosomal locations and most proximategenes 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- Figure 3. ZMPSTE24 transcript levels increase after broadband light (BBL) ment and cellular reprogramming (Gupta et al., 2010; Loewer treatment. (a) Schematic of ZMPSTE24 locus on chromosome 1, hg18. The et al., 2010). Our data provide an initial set of lncRNAs 30-seq (deep sequencing of RNA 30 end) reads were plotted for two old associated with human aging that sets the groundwork for individuals with and without BBL treatment and two untreated young functional studies in the future.
samples. (b) ZMPSTE24 transcript expression is lower in untreated old skin GO analysis of the 151 lncRNAs with significant compared with untreated young skin by quantitative reverse transcription–PCR (RT–qPCR). ZMPSTE24 transcript expression in untreated middle-aged difference in expression between young untreated and aged skin is intermediate (n ¼ 1).
untreated skin showed no significant enrichment for terms.
Similarly, GO analysis of the 42 ‘‘rejuvenated'' lncRNAsshowed no significant enrichment for terms. However, the The NF-kB pathway had been shown to be important in importance of lncRNAs in ‘‘rejuvenation'' is not necessarily skin aging and rejuvenation (Adler et al., 2007), and we diminished. For instance, our 42 lncRNAs are a small number found that the RGs are indeed highly enriched for genes and future studies with greater sample size may identify more bound by NF-kB as measured by chromatin immunoprecipi- lncRNAs, enabling identification of significant GO terms. In tation sequencing experiments. In all, 827 of the 1,293 RGs addition, lncRNAs are a new class of RNA, and our GO are bound by NF-kB (P ¼ 1.2  1075, hypergeometric test).
analysis relied on the proximity of lncRNA sequences to Interestingly, NF-kB itself was not one of the identified RGs.
known genes; it is possible that lncRNAs are important forregulating genes that are not necessarily proximal to the RNA 30 termination appears unaffected by aging or BBL lncRNA (Gupta et al., 2010).
The 3-seq captures the 30 polyadenylated (polyA) RNA The effect of BBL treatment on the immune response fragments for deep sequencing, and thus has the potential includes altering the immune profile in a way that resembles to detect alterations in the location of 30 transcript termina- untreated young skin. Figure 2a shows that although genes tion. The 3-seq method samples RNA sequences immediately related to immune response are ‘‘up'' after treatment, this upstream of the polyA tails. If there were changes in the use ‘‘up'' profile more closely resembles untreated young samples.
of the polyA site within the last exon such that the last exon is This suggests that at least a portion of the immune response lengthened or truncated, this would be detected in the that is ‘‘up'' after treatment is part of the ‘‘rejuvenated'' profile sequencing reads. This method does not evaluate the length and not specific to being treated with BBL.
of the polyA tail.
Journal of Investigative Dermatology (2013), Volume 133 ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL Alternative 30-end usage is an important regulatory Table 1. Top 10 most significantly changed gene mechanism (Mayr and Bartel, 2009), and can alter gene expression levels overall between BBL-treated aged expression output by changing the content of the 30 samples and untreated aged samples untranslated region, which may then alter the repertoire of Fold change: treated aged microRNA targets or RNA-binding proteins (such as those versus untreated aged known to occur in cancer; Shapiro et al., 2011). Thus, inaddition 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 30-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 systemicchanges in mRNA 30 terminations (Figure 4).
Abbreviation: BBL, broadband light.
Treatment-specific effects of BBLIn addition to affecting the age-associated gene expressionprogram, we also considered the possibility that BBL associated skin conditions remains to be seen. Although BBL treatment may induce unique treatment-specific effects that technology has been harnessed for its ability to produce a are distinct from aging. For instance, BBL treatment could more clinically ‘‘youthful'' appearance, our study suggests induce wound healing or scarring response in addition to that ‘‘rejuvenation'' at a molecular level has also occurred, rejuvenation effects. We identified consistent changes in the with a number of genes linked to the aging process being expression of 1,112 genes that occur only in BBL-treated altered in expression after treatment to more closely resemble samples but not in either untreated young or untreated aged young skin. Hence, it is possible that the clinical phenotype samples. Among these treatment-specific genes, the top five represents a functional rejuvenation (at least in the short GO term categories significantly associated with increased term), rather than just a cosmetic mimic of youthful expression after treatment were as follows: immune response (P ¼ 3.8  1012), positive regulation of immune system As the BBL technology has been in existence for o20 process (P ¼ 2.0  108), cell activation (P ¼ 5.7  108), T- years, the long-term effects of BBL remain to be determined.
Although this study assessed the skin 4 weeks after treatment, (P ¼ 1.4  107). These categories are suggestive of an it is unclear how durable the clinical and molecular response immune response to BBL separate from the immune response is. Also unknown is whether there is a decrease of age- genes that are also increased in untreated young samples (as associated skin changes such as seborrheic keratosis or mentioned in the above section). The top five GO term actinic keratosis with time. It may be informative to follow categories significantly associated with decreased expression these current participants in the long term (e.g., 45 years) after treatment were as follows: regulation of transcription with photographs and skin biopsies to determine the duration (P ¼ 2.0  106), transcription (P ¼ 1.7  105), response to of clinical, histologic, and molecular effect of BBL treatment.
organic substance (P ¼ 1.1  104), response to hormone The precise mechanisms by which BBL (noncoherent stimulus (P ¼ 4.4  104), and negative regulation of tran- wavelengths of light) alters gene expression are currently not scription (P ¼ 4.7  104). These genes are distinct from a well understood. For instance, it is known that BBL is previously described ‘‘wound signature'' that characterizes absorbed by different targets including melanin and hemo- response to skin wounding (Chang et al., 2005); however, it is globin, leading to decreased erythema and pigmentation. It is difficult to directly compare signatures with those in our thought that the decrease in fine wrinkling is partly due to the study, as there are no published data at the equivalent time production of new collagen (Fisher et al., 2008). However, point after wounding as used in this study (4 weeks).
the genes identified in this study were not collagen specific. It Finally, the top 10 genes that are most highly upregulated may be possible that if the posttreatment skin biopsies were per- and downregulated in the treated aged samples compared formed earlier than 4 weeks, some of the gene expression changes with the untreated aged samples are listed in Table 1.
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 Our results suggest that regulators of organismal aging can be human kidney and human muscle seem to have distinct altered in human skin using commonly available BBL gene expression signatures (Rodwell et al., 2004; Zahn technology. How such plasticity in aging may be modulated et al., 2006). The aging gene expression profiles of human for healthful benefits such as prevention or treatment of age- skin generated in this study do not appear to be the same as

ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL other reported organ types; however, future direct compar- Adler et al., 2007) and immune response. Our finding that ison studies may shed more light on this issue.
RGs are highly enriched for NF-kB-bound genes suggests that NF-kB is an important regulator of gene expression in BBL may influence pathways controlled by NF-kB. The many contexts. In this case, the most relevant role of NF-kB is precise mechanisms by which this occurs remain to be likely in controlling cell senescence (Bernard et al., 2004; investigated. Nevertheless, our results are consistent with aprior study showing that inducible blockade of NF-kB in agedmurine 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 ofthe disease entity or treatment is not the same as BBL. For instance, gene expression patterns in human postburnhypertrophic scars at 6–15 months in two pediatric and twoadult patients identified six genes as significantly increased Average intensity (Paddock et al., 2003), none of which were significantlychanged in our BBL study. In another example, an in vitrohuman 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 Distance from TSS (bp) 1-month time point when the effects of wound healing mightbe decreasing, and we are more likely to detect rejuvenation effects. At our 1-month time point, NF-kB levels were notsignificantly increased, but genes known to interact withNF-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 rejuvenationeffects and wound repair. In mice and cell culture, thePolycomb pathway controls the lifespan of human fibroblasts(Itahana et al., 2003) and associates with the upregulation ofwound 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 matrixmetalloproteinases 1 and 9 and induce the nuclear transloca-tion of NF-kB after 24–48 hours in human keratinocyteculture (Lee et al., 2009). We did not detect significantincreases in Toll-like receptors 2, 3, and 5, or NF-kB, butour study was in vivo and skin samples were obtained at the1-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 thepopulation of cells within the skin that undergo these changes.
Future studies that get at this question may better explain howBBL treatment might lead to histological or structural changessuch as resorption of elastosis or collagen deposition.
Figure 4. Broadband light (BBL) treatment and aging show no systematicchanges of 30-end usage. (a) Systematic comparison of all 3-seq (deepsequencing of RNA 30 end) reads showing that the majority of reads fell intothe annotated last exon (based on distance of within 1,000 bp fromtranscriptional start site (TSS)) for untreated aged, treated aged, and untreatedyoung groups. The y-axis shows the average intensity of the 3-seq signal.
(b) There were no systematic changes of 30-end usage associated with aging orBBL treatment, as the reads showed similar length distributions between the Distance from transcriptional stop site (kb) untreated aged, treated aged, and untreated young groups.
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 pulse of 10–20 ms duration, with fluences of 8–14 J cm2. At each modalities known to reduce clinical skin aging parameters treatment session, two or more passes were performed. At 4 weeks such as topical tretinoin result in gene expression changes after the third BBL treatment, 4-mm skin biopsies were performed by that are in common with BBL-induced changes.
the Keys punch technique from the treated and adjacent untreated In addition, comparison of non-sun-exposed older skin skin. Punch biopsies (4 mm) were taken from non-sun-exposed arm before and after treatment may identify gene expression skin of five participants o30 years old. These specimens were changes that are specific to intrinsic skin aging.
bisected and placed into either RNAlater (Ambion, cat. no. AM7022, This is an exploratory study, and we will consider Grand Island, NY) or formalin solution for staining with hematoxylin including treated young skin in future studies. In this study, and eosin, von Giesen, or periodic acid–Schiff.
we did not treat younger skin (defined as age o30 yearsfor this study) because there was no clinical indication; The 3-seq and bioinformatics these subjects did not have detectable photoaging or intrinsic Total RNA was extracted using the RNeasy Fibrous Tissue Mini Kit aging on the arm skin. As it is unlikely that BBL would be used (Qiagen, Germantown, MD). The 3-seq was performed as described in in practice on young skin without photoaging (except possibly Beck et al. (2010). In brief, oligo-dT-directed reverse transcription for hair removal), we did not include this group in the study.
generated complementary DNAs corresponding to 30 ends of polyA The current literature on the ability of BBL to induce transcripts; the complementary DNAs were cloned and subjected to collagen neogenesis is contradictory. Although some reports deep sequencing on the Illumina GAIIx (San Diego, CA) platform with on histologic changes induced by BBL include collagen raw read length of 36 bp. Raw reads were aligned to human genome neogenesis (Negishi et al., 2001), there are other studies (hg18) using bowtie (Langmead et al., 2009); each sample generated showing no change (Prieto et al., 2002). This latter study also 6.5–12.4 million uniquely mappable reads. The 30 sequencing of skin reported no change in elastin content after treatment. In our transcripts was performed to assess length distributions.
study, there were no marked changes in collagen content Reads per kilobase of exon per million mappable reads (RPKM, a after treatment on periodic acid–Schiff staining. There were direct measure of transcript abundance) and the number of raw reads decreases in the amount of elastin on von Giesen staining.
falling on to each gene were calculated using a self-developed script We did not detect any significant changes in collagen or by Kun Qu. The Reference Sequence (RefSeq; www.ncbi.nlm.nih.- elastin gene expression levels after treatment. One possibility gov/RefSeq) and Ensembl (http://www.ensembl.org) annotated non- is that the histology was taken at a single time point, and may coding genes were included. Significant genes were called using the not have captured the time when collagen or elastin DESeq package (http://www.bioconductor.org) comparing aged expression levels were more markedly changed. Future treated with aged untreated samples (genes changed because of studies will indeed biopsy-treated skin longitudinally to treatment), and aged untreated with young untreated samples (genes reveal the kinetics of activation/suppression of target genes.
changed because of aging). Unsupervised hierarchical clustering of In addition, it is precisely the goal of this study to extend significantly different expressed genes was performed using Cluster.
beyond the conventional histologic analysis of skin and The GO terms were generated using DAVID (Database for Annotation, explore molecular changes of skin aging and BBL treatment.
Visualization and Integrated Discovery) Bioinformatics Resources 6.7 We observed numerous gene expression changes related to (http://david.abcc.ncifcrf.gov/). Genes close to lncGenes were identified pathways beyond connective tissue organization that can be using GREAT database (http://great.stanford.edu). These data have modulated by BBL.
been deposited into the Gene Expression Omnibus.
Finally, future studies with larger sample size may enable To determine the overlap between RGs and NF-kB binding, we us to identify additional significant genes (both coding and downloaded the NF-kB-bound genes identified by the ENCODE noncoding) whose expression is altered in untreated young project (ENCODE Consortium, 2011) by chromatin immunopreci- versus untreated aged, as well as untreated aged and treated pitation sequencing experiments. A total of 9,650 genes bound NF- aged human skin samples. Larger sample size might also kB in one or more cell types, and these were compared with the RG enable us to correlate the degree of clinical response with more ‘‘rejuvenated'' gene expression changes.
MATERIALS AND METHODS Total RNA was extracted with TRIzol (Invitrogen, Grand Island, NY) Human subjects and sample acquisition followed by RNeasy column purification (Qiagen) and DNAse This study was conducted in accordance with the Declaration of Turbo Treatment (Ambion). RT–qPCR was performed using total Helsinki Principles. After Institutional Review Board approval and RNA (10 ng), Taqman One Step RT–PCR master mix, and one written informed consent was obtained, five female participants over of the following Taqman assays: GAPDH (Hs99999905_m1) and the age of 50 years underwent BBL treatments to the left forearm.
ZMPSTE24 (Hs00956778_m1; Applied Biosystems, Carlsbad, CA).
Inclusion criteria included Fitzpatrick skin type II or III, and a global Reactions were in triplicate for each sample and were performed a assessment of forearm skin aging consistent with moderate or severe minimum of two times. Data were normalized to glyceraldehyde-3- forearm skin aging (modified validated instrument from McKenzie phosphate dehydrogenase (GAPDH) levels.
et al., 2010) for treated participants. Treatments were performed onthe Sciton Joule Platform using the BBL module. The same investigator CONFLICT OF INTEREST performed the treatments at 4-week intervals for a total of three PB has given lectures on broadband light technology. The other authors state treatments using a 515-nm or a 560-nm cutoff filter at a single long no conflict of interest.
ALS Chang et al.
Rejuvenation of Gene Expression in Aging Skin by BBL ment of MAPKs and NFkB in human epidermal keratinocytes. Exp This study was funded by a research grant from Sciton. We are indebted to Dermatol 19:e44–9 Paul Khavari and Jean Tang for prereview of the manuscript. We thank Olena Liang R, Khanna A, Muthusamy S et al. (2011) Post-transcriptional regulation Mykhaylichenko and Sarah Jacobs for administrative support.
of IGF1R by key microRNAs in long-lived mutant mice. Aging cell10:1080–8 SUPPLEMENTARY MATERIAL Liu L, Rando TA (2011) Manifestations and mechanisms of stem cell aging.
J Cell Biol 103:257–66 Supplementary material is linked to the online version of the paper at http://www.nature.com/jid Loewer S, Cabili MN, Guttman M et al. (2010) Large intergenic non-coding RNA-RoR modulates reprogramming of human induced pluripotent stemcells. Nat Genet 42:1113–7 Long X, Spycher C, Han ZS et al. (2002) TOR deficiency in C. elegans causes Adams S, Pankow S, Werner S et al. (2007) Regulation of NFKB activity and developmental arrest and intestinal atrophy by inhibition of mRNA keratinocytic differentiation by the RIP4 protein: implications for translation. Curr Biol 12:1448–61 cutaneous wound repair. J Invest Dermatol 127:538–44 de Magalhaes JP, Wuttke D, Wood SH et al. (2012) Genome-environment Adler AS, Sinha S, Kawahara TLA et al. (2007) Motif module map reveals interactions that modulate aging: powerful targets for drug discovery.
enforcement of aging by continual NFKB activity. Genes Dev 21:3244–57 Pharmacol Rev 64:88–101 Beck AH, Weng Z, Witten DM et al. (2010) 30-end sequencing for expression Mayr C, Bartel DP (2009) Widespread shortening of 30UTRs by alternative quantification (3SEQ) from archival tumor samples. PLoS One 19:5: cleavage and polyadenylation activates oncogenes in cancer cells. Cell Bernard D, Gosselin K, Monte D et al. (2004) Involvement of Rel/ NFkB McKenzie NE, Saboda K, Duckett LD et al. (2010) Development of a transcription factors in keratinocyte senescence. Cancer Res 64:472–81 photographic scale for consistency and guidance in dermatologic Bitter Jr PH (2000) Noninvasive rejuvenation of photodamaged skin using assessment of forearm sun damage. Arch Dermatol 147:31–6 serial, full-face intense pulsed light treatments. Dermatol Surg 26:835–43 Nelson TJ, Behfar A, Yamada S et al. (2009) Stem cell platforms for Caslini C, Connelly JA, Serna A et al. (2009) MLL associates with telomeres regenerative medicine. Clin Transl Sci 2:222–7 and regulates telomeric repeat-containing RNA transcription. Mol Cell Negishi K, Tezuka Y, Kushikata N et al. (2001) Photorejuvenation for Asian Biol 29:4519–26 skin by intense pulsed light. Dermatol Surg 27:627–31 Chang HY, Nuyten DS, Sneddon JB et al. (2005) Robustness, scalability and Paddock HN, Schultz GS, Baker HV et al. (2003) Analysis of gene expression integration of a wound-response gene expression signature in predicting patterns in human postburn hypertrophic scars. J Burn Care Rehabil 24:371–7 breast cancer survival. Proc Natl Acad Sci USA 102:3738–43 Parado J, Bougria M, Ayala A et al. (1999) Effects of aging on the various steps Chen C, Liu Y, Liu Y et al. (2009) mTOR regulation and therapeutic rejuve- of protein synthesis: fragmentation of elongation factor 2. Free Radic Biol nation of aging hematopoietic stem cells. Sci Signal 2:p.ra71 Curran SP, Ruvkun G (2007) Lifespan regulation by evolutionarily conserved Partridge L (2010) The new biology of ageing. Phil Trans R Soc B 12 365:147–54 genes essential for viability. PLoS Genet 3:e56 Prieto VG, Sadick NS, Lloreta J et al. (2002) Effects of intense pulsed light on ENCODE Consortium (2011) The user's guide to the encyclopedia of DNA sun-damaged human skin, routine, and ultra-structural analysis. Lasers elements. PLoS Biol 9:1001046 Surg Med 30:82–5 Fisher GJ, Varani J, Voorhees JJ (2008) Looking older: fibroblast collapse and Rodwell G, Sonu R, Zahn JM et al. (2004) A transcriptional profile of aging in therapeutic implications. Arch Dermatol 144:666–72 the human kidney. PLoS Biol 2:e427 Greer EL, Maures TJ, Hauswirth AG et al. (2010) Members of the histone H3 Serrano M (2011) Cancer: final act of senescence. Nature 479:481–2 lysine 4 trimethylation complex regulate lifespan in germline-dependent Shapiro IM, Cheng AW, Flytzanis NC et al. (2011) An EMT-driven alternative manner in C. elegans. Nature 466:383–7 splicing program occurs in human breast cancer and modulates cellular Gupta RA, Shah N, Wang KC et al. (2010) Long non-coding RNA HOTAIR phenotype. PLoS Genet 7:e1002218 reprograms chromatin state to promote cancer metastasis. Nature 464: Shaw T, Martin P (2009) Epigenetic reprogramming during wound healing: loss of polycomb-mediated silencing may enable upregulation of repair Hsieh C-C, Kuro-o M, Rosenblatt KP et al. (2010) The ASK1-Signalosome genes. EMBO Rep 10:881–6 regulates p38 MAPK activity in response to levels of endogenous Tariq M, Kim HJ, Jejelowo O et al. (2011) Whole-transcriptome RNAseq oxidative stress in the Klotho mouse models of aging. Aging 2:597–611 analysis from minute amount of total RNA. Nucl Acids Res 39:e120 Hwang JS, Hwang JS, Chang I et al. (2007) Age-associated decrease in Tazearslan C, Huang J, Barzilai N, Suh Y (2011) Impaired IGF1R signaling proteasome content and activities in human dermal fibroblasts: in cells expressing longevity-associated human IGF1R alleles. Aging cell restoration of normal level of proteasome subunits reduces aging markers in fibroblasts from elderly persons. J Gerontol A Biol Sci MedSci 62:490–9 Wang Z, Gersten M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63 Itahana K, Zou Y, Itahana Y et al. (2003) Control of the replicative life span of human fibroblasts by p16 and the polycomb protein Bmi-1. Mol Cell Biol Wapinski O, Chang HY (2011) Long noncoding RNAs and human disease.
Trends Cell Biol 21:354–61 Kirby K, Hu J, Hilliker AJ et al. (2002) RNA interference-mediated Zahn JM, Sonu R, Vogel H et al. (2006) Transcriptional profiling of silencing of Sod2 in Drosophila leads to early adult-onset mortality aging in human muscle reveals a common aging signature. PLoS Genet and elevated endogenous oxidative stress. Proc Natl Acad Sci USA Langmead B, Trapnell C, Pop M et al. (2009) Ultrafast and memory-efficient This work is licensed under the Creative Commons alignment of short DNA sequences to the human genome. Genome Biol Unported License. To view a copy of this license, visit Lee Y, Kim H, Kim S et al. (2009) Activation of toll-like receptors 2, 3, or 5 induces matrix metalloproteinase-1 and -9 expression with the involve- Journal of Investigative Dermatology (2013), Volume 133

Source: http://sciton.com.ua/images/files/Rejuvenation_of_Gene_Expression_by_BBL_lq.pdf