Research from Stanford University has shown that the Forever Young BBL procedure can rejuvenate aging skin cells at the gene level, making them functionally similar to young skin cells.
Research carried out on model organisms suggests that senescent cells can be functionally rejuvenated. But does this concept apply to human skin? Until now, this question has remained unproven. We invite you to familiarize yourself with the results of a study in which the gene changes occurring as a result of photo and natural aging (chrono aging) of human skin, as well as the effect of broadband light (BBL) procedures on these processes, were studied. It turned out that skin aging is associated with significant changes in RNA and that BBL procedures allow them to "rejuvenate", that is, to make them more similar in expression to the genes of young skin cells. Thus, BBL procedure can restore gene expression in cells with photo- and chronological aging of human skin, similar to cells of young skin.
* RNA, ribonucleic acids, are molecules that carry information from the genome represented by DNA (deoxyribonucleic acid). In genome (DNA) research, RNA molecules are studied as molecules that are carriers of information for certain sections of DNA. Therefore, the reference to RNA hereinafter describes the processes occurring in DNA, i.e. in the genome of cells. (Note).
Anne Lynn S. Chang, Stanford University Department of Dermatology, School of Medicine (Redwood City, CA, USA)
Patrick H. Bitter Jr, Department of Aesthetic Dermatology (Los Gatos, CA, USA)
KunQu, Stanford University Department of Dermatology, School of Medicine (Redwood City, CA, USA)
Meihong Lin, Stanford University Department of Dermatology, School of Medicine (Redwood City, CA, USA)
Nicole A. Rapicavoli, Department of Dermatology, Stanford University, Howard Hughes Medical Institute (Stanford, CA, USA)
Howard Y. Chang, Department of Dermatology, Stanford University, Howard Hughes Medical Institute (Stanford, CA, USA)
Published: J Invest Dermatol. 2013 Feb; 133(2): 394–402.
Aging is a complex process of complex genetic and environmental changes and is associated with multiple changes in gene expression. But how changes at the genetic level affect human health is still unknown. Numerous mutations of single genes show life extension in model organisms (Partridge, 2010; de Magalhaes et al., 2012). Diet restrictions can also slow the rate of aging, even when used late in life (Partridge, 2010). More recently, several presentations have shown the rejuvenation of senescent cells or tissues, demonstrating the amazing reversibility of aging. For example, heterochronous parabiosis of young and old mice releases circulatory factors that restore the function of senescent muscle stem cells (Liu and Rando, 2011). In addition, blocking the transcription factor NF-kB in old cells of the mouse epidermis can interrupt cellular senescence and restore the overall gene expression of old skin cells, making them similar to young cells (Adler et al., 2007).
The question arises: do the aging processes in human skin, which develop over decades, and not months or years, as was shown in mouse models, have the same reversibility? Identifying life-saving methods of blocking aging is a major challenge.
Broadband light (BBL), also known as broadband pulsed light, a widely available and popular skin “rejuvenation” procedure, is the ideal technology to test this concept. According to the American Society for Aesthetic and Plastic Surgery (ASAPS), in 2009 more than $ 215 million were spent on these procedures in the United States. Unlike ablation procedures, which improve skin condition through thermal destruction and regeneration of the epidermis and the superficial dermis, BBL uses broadband incoherent light in the range from 560 to 1,200 nm, which is absorbed by many skin components. Currently, BBL procedures are used to reduce the severity of wrinkles, pigmentation disorders, erythema and elastosis (Bitter Jr, 2000; Negishi et al., 2001). However, the molecular changes caused by this procedure have not yet been described.
The term "rejuvenation" is used by both researchers and the general population, and everyone puts their own concept into it. That is why it is required to decide on a definition. In this study, we interpret the concept of "rejuvenation" as the restoration of youth indicators in aging cells and tissues. Is the skin really about after the BBL procedure rejuvenates at the molecular level and becomes similar to young skin, or are the changes taking place associated with wound processes and damage regeneration, which is fundamentally different from young, intact skin?
Histological studies confirm that BBL significantly reduces melanin in the dermis and eliminates telangiectasias (Bitter Jr, 2000; Prieto et al., 2002).
Several studies have also reported an increase in collagen in the upper layers of the papillary dermis by the third week after the procedure (Negishi et al., 2001). However, the synthesis of new collagen can have a variable or short-term effect: data from ultrastructural analysis of the skin 3 months after the procedure showed no signs of an effect on collagen and elastin (Prieto et al., 2002).
We have studied the molecular basis of response to BBL procedures by determining the overall gene expression of human skin with photo and chronological aging after BBL procedures. The aim was to track a wide range of changes in the RNA of senescent cells under the influence of BBL, including changes in gene expression (encoded and non-encoded) and gene regulation.
Study participants and materials
This study was conducted in accordance with the Declaration of Helsinki. After reading and signing informed consent, five female participants over the age of 50 underwent BBL on the left forearm. Inclusion criteria were Fitzpatrick skin type II or III and moderate to severe aging of the forearm skin (McKenzie et al., 2010). Tanning salons, topical retinoids, and any hand treatments were prohibited 1 month prior to and throughout the study. Participants were instructed to protect their hands with a wide range of sunscreens and long-sleeved clothing, and to avoid unnecessary sun exposure.
Procedures were performed on Sciton's Joule platform using the BBL module. Participants underwent 3 procedures with an interval of 4 weeks using 515 or 560 nm filters with one pulse of 10–20 ms duration with a specific power of 8–14 J/cm2. Two or more passes were performed during one procedure.
4 weeks after the 3rd BBL procedure, punch biopsies of 4 mm skin areas were performed on the treated skin and on the adjacent untreated area. Punch biopsies (4 mm) were also performed from skin areas not exposed to sunlight in five participants under the age of 30 who had the same criteria for inclusion in the study, but did not show signs of photoaging on the hands.
Samples were halved and placed in RNA stabilization solution or formalin solution for hemotoxylin-eosin staining.
Clinical and histological changes
After three BBL procedures on the skin of the hands, there was an improvement in the clinical indicators of chronological aging and aging of the skin under the influence of environmental factors: fine wrinkles (P = 0.03), pigmentation (P = 0.02) and general indicators of skin aging (P = 0.01)
(Fig. 1 a – c). According to histological examination, signs of elastosis in aging skin samples from BBL sites were less pronounced than in aging skin samples. Skin not exposed to BBL (Fig. 1 d – g). PAS staining of histological specimens showed no change in the amount of collagen in the dermis between treated and untreated skin samples, although collagen fibers were less disaggregated after the BBL procedure (Fig. 1 h – i). Samples of aging skin after the procedure showed a subjective increase in epidermal density (Fig. 1 e, g, i) compared to samples of aging skin that were not exposed (Fig. 1 d, f, h).
Genetic Alteration Control
To pinpoint the signs of aging at the ends of RNA molecules, we first identified transcriptional abnormalities in gene regions associated with aging by comparing young skin samples with untreated aging skin samples, and then tested the effect of the BBL procedure on changing these parameters.
Comparison of mRNA transcription levels in young untreated skin and aging untreated skin, as well as in aging untreated and aging treated skin showed a significant increase in expression levels in 3,530 genes (Fig. 2a). The direction of gene changes after the BBL procedure is shown in Figure 2a: a twofold decrease in expression is shown in blue, and a twofold increase in expression in yellow. For a visual display of significant genes, we used a visual and heat map. To the right of it, we have placed a table, the purple stripes of which represent the main biological effects for which genes are responsible, in accordance with the gene ontology. For example, "rejuvenated genes" and lncRNAs (long non-encoded RNAs) are located at both the top and bottom of the visual heatmap. The genes responsible for the immune response and translation are located in the lower half of the map. The genes responsible for cell adhesion are located at the top of the map. The activity of these genes is decreased in the group of young volunteers and increased in the group of untreated volunteers with aging skin, and in the group of treated volunteers with aging skin, the activity of adhesion genes is at an average level. The adjacent table with purple columns indicates which genes are changing and for which biological processes these genes are responsible. A visual heat table using "warm" (yellow - increase) and "cold" (blue - decrease) colors indicates the direction of changes in gene activity - an increase or decrease in their function, which allows you to compare young skin, aging untreated and aging treated skin. For example, young skin and treated old skin show increased levels of transcription, which are responsible for "immune response" and "translation", as both groups showed increased gene activity (yellow). And vice versa: in the group of untreated patients with aging skin, there is a decrease in the activity of genes (blue) "immune response" and "translation" in comparison with the other two groups.
The gene changes associated with aging are multifaceted and combine changes in several biological functions.
The first five most pronounced changes with increased gene activity in aging untreated skin in comparison with young untreated skin include:
• translation (P = 4.7 x 10-12);
• translational lengthening (P = 5.1 x 10-7);
• macromolecular complex complexes (P = 7.5 x 10-6);
• ncRNA (non-encoded RNA) responsible for metabolism (P = 6.2 x 10- 6);
• RNA processing (P = 2.5x10-6).
The first five genes with decreased function in the group of aging untreated skin in comparison with young skin included:
• genes responsible for cell adhesion (P = 1.5 x 10-17);
• biological adhesion ( P = 1.5 x 10-17);
• homophilic cell adhesion (P = 7.8 x 10-8);
• gene responsible for skeletal development (P = 3.2 x 10-7 );
• enzyme-linked protein-receptor signaling pathway (P = 5.2 x 10-6).
Changes in these genes resemble changes in the set of genes during aging in other tissues and organisms.
BBL contributes altered gene expression Genes whose expression level in aging skin treated with BBL was closer to that of young untreated skin were designated RGs ("rejuvenated genes").
Hierarchical clustering showed that gene expression of treated aging skin was more similar to untreated skin younger skin than untreated aging skin (Fig. 2 a). Rejuvenated genes are responsible for certain biological functions linked to them, the six most pronounced of them are:
• translation (P = 5.8 x 10-11);
• RNA processing (P = 6.3 x 10-8);
• ncRNA-processing of metabolism (P = 1.4 x 10-7);
• regulation of cellular protein metabolism (P = 1.6 x 10-5);
• macromolecular catabolic processes (P = 2.1 x 10-5);
• cell cycle (P = 2.4 x 10-5) (Fig. 2 b, top right).
A more detailed study showed the expression " genes rejuvenated by the BBL procedure linked to key regulators known as genes that control aging (Fig. 2c).
• ZMPSTE24 - a metalloproteinase that processes lamami Mr. A. A defect in this gene causes the syndrome of severe premature aging, Hutchinson-Guildford progeria;
• IGF1R receptor - this gene is directly associated with aging and longevity in models of humans, mice and other organisms (Liang et al., 2011; Tazearslan et al., 2011);
• other “rejuvenated genes” - EIF4G1 and EIF4EBP1 - are responsible for increasing lifespan in Caenorhabditis elegans (a free-living nematode that is widely used as a model organism) (Curran and Ruvkun, 2007);
• MLL is a telomere-associated transcriptional regulator (Caslini et al., 2009), and H3K4 methylate, which is responsible for normal lifespan in C. elegans (Greer et al., 2010);
• MAP3K5 (ASK10) - regulates the activity of kinases in response to oxidative stress in the Klotho model of aging mice (Hsieh et al., 2010);
• PSMD8 - is a component of proteasomes, the disruption of which contributes to the aging of human skin (Hwang et al., 2007); < br>• RING1 and MOV10 - part are involved in processes that control the lifespan of human fibroblasts (Itahana et al., 2003);
• EEF2 (eukaryotic elongation translation factor 2) - was also classified as a "rejuvenated genes". Research has shown it is associated with decreased protein synthesis in rats (Parado et al., 1999);
• Finally, a number of suppressor genes that control the cell cycle and ensure genome integrity, such as ING4, tumor suppressor, DAXX and MSH2 were also "rejuvenated." Figure 2a shows that although genes for the immune response “increase” activity after the procedure, this “enhancement” profile is more reminiscent of young, untreated skin. This suggests that the small part of the immune response that "increased" after the procedure is part of the "rejuvenation" and is a non-specific response to the Forever Young BBL procedure.
Thus, the Forever Young BBL procedure is able to restore many of the molecular properties of young skin in aging human skin cells, at least in the short term. In addition, we did not find changes in gene expression associated with wound processes and scar formation.
Specific response to Forever Young BBL procedure
We also considered the possibility that the BBL procedure may have specific therapeutic effects not related to aging. For example, Forever Young BBL could stimulate wound healing or scar formation in addition to rejuvenating effects.
We found significant changes in the expression of 1,112 genes that were only seen in samples that underwent the Forever Young BBL rejuvenation, but these changes were not detected in untreated young and untreated aging skin samples. Among these genes with a specific response to the procedure, five most pronounced biological effects were identified with an increase in gene expression after the procedure:
- immune response (P = 3.8 x 10-12);
- positive regulation of the immune system (P = 2.0 x 10-8);
- cell activation (P = 5.7 x 10-8);
- activation of T-cells (P = 6.0 x 10-7);
- defensive reactions (P = 1.4 x 10-7).
These reactions are associated with the immune response to Forever Young BBL rejuvenation and differ from the immune response of genes that were also increased in untreated youngsters. The five most significant biological effects associated with decreased gene expression after the procedure were:
- regulation of transcription (P = 2.0 x 10-6);
- transcription (P = 1.7 x 10-5);
- response to organic matter (P = 1.1 x 10-4);
- response to hormonal stimuli (P = 4.4 x 10-4);
- negative regulation of transcription (P = 4.7 x 10-4).
These genes differ from previously described “healing” effects that characterize skin healing processes (Chang et al., 2005), but it is difficult to make a direct comparison, since there is no published data equivalent to ours on the activity of genes associated with wound processes on the 4th week.
And finally, the top 10 genes that increase and decrease activity in treated BBL with aging skin in comparison with untreated aging skin are given in Table 1.
Our results show that the body's aging regulators can be altered in human skin through the use of publicly available BBL technology. How plastic aging processes are and how much they can change towards healthier skin remains to be seen.
Although BBL technology is used more to give skin a "youthful" appearance, our research has shown that skin rejuvenation is observed at the molecular level, with numerous changes in the work of genes associated with the aging process; that the visible clinical changes are due to functional rejuvenation of the life (at least for a short period of time), rather than an external cosmetic imitation of the appearance of young skin.
Since BBL technology has been around for less than 20 years, the long-term effects of BBL therapy have yet to be determined. Although this study assessed the condition of the skin 4 weeks after the procedure, it is unclear how long this clinical effect and molecular changes persist. It is also unknown how persistently the effect persists in age-related conditions such as seborrheic or actinic keratosis over time. It may be informative to investigate current participants in the long term, with photographs and skin biopsies to determine the duration of the clinical, histological and molecular effects of the Forever Young BBL skin rejuvenation procedure.
Exact mechanisms by which BBL (Incoherent Light Radiation) alters expression genes are currently not well understood. For example, it is known how BBL is absorbed by various targets - melanin and hemoglobin, which leads to a decrease in erythema and pigmentation.
This is preliminary research and we will study the effect of BBL on young skin. In these trials, we did not perform BBL procedures on the skin of persons younger than 30 years old, since they did not have clinical indications - distinguishable signs of photo- and chrono-aging of the skin of the forearms. Since it is unlikely that BBL will be used in practice on young skin without signs of photoaging (except for hair removal), we did not include this group in the study.
Data on the possible effect of BBL on collagen synthesis are conflicting, according to available sources. Although some reports indicate histological changes induced by the BBL procedure, including collagen neogenesis (Negishi et al., 2001), other studies have not noted these changes (Prieto et al., 2002). According to the latest study, there was also no change in elastin content after the procedure. In our experiments, there were no significant changes in collagen content after the procedure on PAS-stained samples. They had a decrease in the amount of elastin on the von Giesen stained slides. We found no significant changes in collagen and elastin gene expression levels after the procedure. Perhaps the reason lies in one collection of materials for tests, which could not capture the period of time when the level of expression of collagen and elastin genes underwent significant changes.
Subsequent studies with skin biopsy will reveal the kinetics of activation/suppression of target genes. In addition, the aim of this study was to go beyond simple histological analysis of the skin and study the molecular changes during skin aging and the effect on them of Forever Young BBL skin rejuvenation procedures. We have investigated the many changes in connective tissue genes that can be modulated by BBL.
Subsequent studies with large samples may allow us to identify additional significant genes (both encoded and non-encoded) whose expression changes in comparison to aging untreated skin with young untreated skin as well as untreated aging skin with treated aging skin. Large samples may allow correlating the degree of clinical response with changes in the "rejuvenated" genes.
Before & After photo
Before and after photos from previous research by Dr. Patrick Bitter "Effects of BBL on the Skin in the Long Term".