Augmentation of Skin Aging Reversal By Broadband Light With Skin Tightening Properties Via Gene Expression Analysis
Phase: NA
Conditions: Aging
Sponsor: Stanford University

 
 
 
 
 
Protocol title: 
 
Infrared and Broadband Light for Skin Aging 
NCT03243981 
 
Version date 06162016 
 
 
Principal Investigator: 
 
Anne Lynn S. Chang, MD 
Assistant Professor of Dermatology 
Stanford University School of Medicine 
450 Broadway Street, MC 5334 
Redwood City, CA 94305 
Phone 650 644 7318 
alschang@stanford.edu 
 
 
Co-investigator: 
 
 
Patrick Bitter, MD 
Tyler Hollmig, MD 
 
Sponsor: 
 
Sciton 
 
 
 
 

Primary Objectives 
 
-To assess the effect of broadband light with skin tightening properties on the 
gene expression pattern of aging skin 
 
-To assess the duration of the skin rejuvenation effects of broadband light with 
skin tightening properties (optional) 
 
Background 
 
 
Studies in model organisms suggest that aged cells can be functionally 
rejuvenated, but whether this concept applies to human skin is unclear. Recently, 
we applied 3'-end sequencing for expression quantification ("3-seq") to discover 
the gene expression program associated with human photoaging and intrinsic 
skin aging (collectively termed "skin aging"), and the impact of broadband light 
(BBL) treatment. We found significant changes in 2,265 coding and noncoding 
RNAs, of which 1,293 that became “rejuvenated” after BBL treatment, whereby 
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. Hence, BBL treatment can restore gene 
expression pattern of photo-aged and intrinsically aged human skin to resemble 
young skin.  
 
However, the duration of these effects and the potential to augment these 
effects through increases in particular wavelengths of light have not been 
explored. The Sciton SkinTyte (800-1800nm) is the ideal technology to examine 
these questions, since this device has been used in the clinical setting to reduce 
cheek and submental laxity (Gold, 2010). It incorporates the broadband light 
technology with an emphasis on 590 nm filter to achieve these clinical results.  
 
 
Study Procedures 
 
The design of this study is a prospective, single blind study of six adult 
volunteers. 
Human subjects and sample acquisition 
This study will be conducted in accord with Declaration of Helsinki 
principles. After Institutional Review Board approval and informed consent is 
obtained, six female participants over the age of 55 years will undergo BBLST 
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., 
2011) for treated participants. Treatments will be performed on the Sciton Joule 
Platform using BBL in Skintyte mode with 590ST filter. On a separate part of the 
arm that is clearly marked, Skintyte alone will be applied. Untreated areas will 
also be marked. All markings will be photographed. The same investigator will 
perform the treatments at 4-week intervals for a total of 3 treatments.  At each 

treatment session, two or more passes were performed. Four weeks after the 
third BBL treatment, 4 mm skin biopsies will performed by Keys punch technique 
from the BBLST treated, ST treated and adjacent untreated skin. These 
specimens will be bisected and placed into either RNAlater (Ambion Cat# 
AM7022) or formalin solution for with H&E, von Giesen or PAS staining. 
Inclusion criteria 
1. Female 
2. Age 55 years or older (total 6)  
3. Fitzpatrick skin type 2-3 
4. Photo-aging at least moderate on the extensor forearms 
 
Exclusion criteria 
 
1. Unable to understand and sign informed consent form 
2. Unable to comply with study procedures 
3. Pregnant or lactating 
4. Prior treatment to forearms including topical retinoid, laser treatment, 
photodynamic treatment, prescription topical agents x 1 month 
5. Active skin conditions that precluding treatment including zoster, blistering 
skin disease, psoriasis, atopic dermatitis, abnormal scarring, skin cancer 
in the area of study treatment 
6. Currently on hormone based therapy (both systemic and topical) 
 
 
Screening visit  
 
After written informed consent signing, subjects will undergo the following 
procedures, eligibility will be assessed and the following will be obtained: 
 
 Demographics 
 Medical Histories 
 Physical Examination 
 Photographs with high resolution digital camera 
 Cutometry 
 
Baseline (visit 1, treatment #1), Day 28 visit (treatment #2), Day 56 visit 
(treatment #3) 
 
Each subject would have three treatments at one month intervals by the same 
health care provider. Each subject would have three biopsies at approximately 
one month post treatment (day 84 visit). The biopsies will be of the BBL treated, 
the BBL-ST treated and the untreated. Total biopsy per patient at the end of the 
study is 3. 
 
 

 
Clinical characteristics 
Mean ages, skin type and medical histories will be described. The mean elasticity 
and transepidermal water loss will be described for treated (day 84 and possibly 
168), untreated (day 84 or day 168 (if we go with the latter budget scenario)). 
The degree of fine wrinkling, sagging, texture, erythema and overall appearance 
will be assessed using a 5-point Likert scale and compared for treated, untreated 
and young skin. T-tests will be used to assess the mean, standard deviations and 
p values at a significance level <0.05 as described in Chang et al., 2013. 
 
3-Seq and bioinformatics 
Total RNA will be extracted using RNeasy Fibrous Tissue Mini Kit 
(Qiagen). 3-seq will be performed as described in Beck AH et al., 2010. In brief, 
oligo-dT-directed reverse transcription generated cDNAs corresponding to 3' 
ends of poly-adenylated transcripts; the cDNAs were cloned and subjected to 
deep sequencing on the Illumina GAIIx platform with raw read length 36bp. 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. 3’ 
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 will be calculated using self-developed script by K. Qu.  RefSeq 
(www.ncbi.nlm.nih.gov/RefSeq) and Ensembl (http://www.ensembl.org) 
annotated noncoding genes were included. Significant genes will be called using 
DESeq package (http://www.bioconductor.org ) comparing aged-treated versus 
aged-untreated samples (genes changed due to treatment), and aged-untreated 

versus young-untreated (genes changed due to aging). Unsupervised 
hierarchical clustering of significantly different expressed genes will be performed 
using Cluster. Treated versus untreated skin across the time points of Study Day 
28 and Day 56 will analyzed. The young group will serve as the comparison 
group. 3’-seq technique will allow for analysis of long non-coding RNAs. 
Gene Otology terms were generated using DAVID Bioinformatics 
Resources 6.7 (http://david.abcc.ncifcrf.gov/). Genes close to lncGenes will 
identified using GREAT database (http://great.stanford.edu).  This data will be 
deposited into the Gene Expression Omnibus (GEO). 
RT-qPCR  
Total RNA will be extracted with TRIzol (Invitrogen) 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). Reactions will be in 
triplicate for each sample and performed a minimum of two times. Data will be 
normalized to GAPDH levels. 
 
Study Endpoints 
 
-effect of broadband light with skin tightening properties on the gene expression 
pattern of aging skin as measured by 3’-end sequencing, between treated and 
untreated 
 
-duration of the skin rejuvenation effects of broadband light with skin tightening 
properties including cutometry, transepidermal water loss, sagging, fine wrinkling, 
coarse wrinkling and physician global assessment between pre-treatment, day 
84 (one month after last treatment) (and possibly day 156 (2 months after last 
treatment) if budget allows). 
 
References 
Adams S, Pankow S, Werner S, Munz B (2007). Regulation of NFKB activity and 
keratinocytic differentiation by the RIP4 protein: implications for cutaneous 
wound repair. JID 127(3): 538-44.  
Adler AS, Sinha S, Kawahara TLA, et al. (2007). Motif module map reveals 
enforcement of aging by continual NFKB activity. Genes and Dev 21:3244-57. 
Beck AH, Weng Z, Witten DM, et al. (2010). 3’-end sequencing for expression 
quantification (3SEQ) from archival tumor samples. PLoS One 19; 5(1): e8768. 
Bernard D, Gosselin K, Monte D et al. (2004) Involvement of Rel/ NFkB 
transcription factors in keratinocyte senescence. Cancer Res 64: 472-81. 

Caslini C, Connelly JA, Serna A, et al. (2009). MLL associates with telomeres 
and regulates telomeric repeat-containing RNA transcription. Mol Cell Biol 29 
(No. 16): 4519-4526. 
Chang A, Bitter PB et al. (2013) Rejuvenation of gene expression pattern of aged 
human skin by broadband light. J of Investig Dermatol 133(6): 1691. 
Chang HY, Nuyten DS, Sneddon JB et al. (2005). Robustness, scalability and 
integration of a wound-response gene expression signature in predicting breast 
cancer survival. PNAS USA 102(10): 3738-3743. 
Chen C, Liu Y, Liu Y, Zheng P. (2009) mTOR regulation and therapeutic 
rejuvenation of aging hematopoietic stem cells. Sci Signal. 2(98): p.ra71. 
Curran SP, Ruvkun G (2007). Lifespan regulation by evolutionarily conserved 
genes essential for viability. PLoS Genet 3(4):e56. 
ENCODE Consortium (2011). The user’s guide to the encyclopedia of DNA 
elements. PLoS Biology 9(4): 1001046. 
Fisher GJ, Varani J, Voorhees JJ (2008). Looking older: fibroblast collapse and 
therapeutic implications. Arch Dermatol 144 (5). 
Gold MH (2010). Update on tissue tightening. Journal of clinical and aesthetic 
dermatology. 3(5). 
Goldberg DJ (2000). New collagen formation after dermal remodeling with an 
intense pulsed light source. J Cutan Laser Ther 2:59-61.  
Greer EL, Maures TJ, Hauswirth AG, et al. (2010). Members of the histone H3 
lysine 4 trimethylation complex regulate lifespan in germline-dependent manner 
in c. elegans. Nature 466(7304):383-387. 
Gupta RA, Shah N, Wang KC, et al. (2010). Long non-coding RNA HOTAIR 
reprograms chromatin state to promote cancer metastasis. Nature 464:1071-
1076. 
Holzenberger M, Dupont J, Ducos B et al. (2003). IGF1 receptor regulates 
lifespan and resistance to oxidative stress in mice. Nature 421:182-185. 
Hsieh C-C, Kuro-o M, Rosenblatt KP, et al. (2010). The ASK1-Signalosome 
regulates p38 MAPK activity in response to levels of endogenous oxidative stress 
in the Klotho mouse models of aging. Aging 2(9):597-611. 

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 23 (No. 
1):389-401. 
Kirby K, Hu J, Hilliker AJ, Phillips JP (2002). RNA interference-mediated 
silencing of Sod2 in Drosophila leads to early adult-onset mortality and elevated 
endogenous oxidative stress. PNAS 99(25): 16162-7. 
Langmead B, Trapnell  C, Pop M, et al. (2009). Ultrafast and memory-efficient 
alignment of short DNA sequences to the human genome.  Genome Biology 
10:R25 
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 involvement of 
MAPKs and NFkB in human epidermal keratinocytes. Experimental Dermatology, 
19: e44-49. 
Liu L, Rando TA (2011). Manifestations and mechanisms of stem cell aging. JCB 
103 (No. 2):257-266. 
Loewer S, Cabili MN, Guttman M, et al. (2010). Large intergenic non-coding 
RNA-RoR modulates reprogramming of human induced pluripotent stem cells. 
Nature Genetics 42:1113-1117. 
Long X, Spycher C, Han ZS, et al. (2002). TOR deficiency in C. elegans causes 
developmental arrest and intestinal atrophy by inhibition of mRNA translation. 
Curr Biol 12: 1448-61. 
de Magalhaes JP, Wuttke D, Wood SH, et al. (2012). Genome-environment 
interactions that modulate aging: powerful targets for drug discovery. Pharmacol 
Rev 64(1):88-101. 
Mayr C, Bartel DP (2009). Widespread shortening of 3’UTRs by alternative 
cleavage and polyadenylation activates oncogenes in cancer cells. Cell 
138(4):673-84. 
McKenzie NE, Saboda K, Duckett LD et al. (2010). Development of a 
photographic scale for consistency and guidance in dermatologic assessment of 
forearm sun damage. Arch Dermatol 147 (1): 31-36. 
 
Mehler MF, Mattick JS (2007). Noncoding RNAs and RNA editing in brain 
development, functional diversification, and neurological disease. Physiol Rev 87 
(No. 3):799-823. 
 

Morozova O, Hirst M, and Marra MA (2009). Applications of new sequencing 
technologies for transcriptome analysis. Annu Rev Genomics Hum Genet 
10:135-51. 
 
Nelson TJ, Behfar A, Yamada S, et al. (2009). Stem cell platforms for 
regenerative medicine. Clinical and Translational Science 2(3):222-227. 
Negishi K, Tezuka Y, Kushikata N, et al. (2001). Photorejuvenation for Asian Skin 
by Intense pulsed light. Dermatol Surg 27:627-31.  
Paddock HN, Schultz GS, Baker HV et al. (2003). Analysis of gene expression 
patterns in human postburn hypertrophic scars, J Burn Care Rehabil 24(6): 371-
7. 
 
Parado J, Bougria M, Ayala A, et al. (1999). Effects of aging on the various steps 
of protein synthesis: fragmentation of elongation factor 2. Free Radic Biol Med 
26:362-370. 
 
Partridge L (2010). The new biology of ageing. Phil Trans R Soc B 12 365 (No. 
1537): 147-154. 
Prieto VG, Sadick NS, Lloreta J, et al. (2002). Effects of intense pulsed light on 
sun-damaged human skin, routine, and ultra-structural analysis. Lasers Surg 
Med 30:82-5.  
Rodwell G, Sonu R, Zahn JM, et al. (2004).  A transcriptional profile of aging in 
the human kidney. PLoS Biology 2(12). 
Serrano M (2011). Cancer: final act of senescence. Nature 479, 481-482. 
Shapiro, IM, Cheng, AW, Flytzanis, NC et al. (2011). An EMT-driven alternative 
splicing program occurs in human breast cancer and modulates cellular 
phenotype. PLoS Genet. 7, e1002218. 
Shaw T, Martin P (2009) Epigenetic reprogramming during wound healing: loss 
of polycomb-mediated silencing may enable upregulation of repair genes. EMBO 
reports 10 (8): 881-6 
Tariq M, Kim HJ, Jejelowo O, et al. (2011). Whole-transcriptome RNAseq 
analysis from minute amount of total RNA. Nucl. Acids Res. 
Wang Z, Gersten M, Snyder M (2009). RNA-Seq: a revolutionary tool for 
transcriptomics. Nat Rev Genet 10(1): 57-63. 
Wapinski O, Chang HY (2011). Long noncoding RNAs and human disease. 
Trends in Cell Biology 21(6): 354-61.