WO2012151346A1

WO2012151346A1 - Expression signatures of genes and gene networks associated with skin aging

Info

Publication number: WO2012151346A1
Application number: PCT/US2012/036226
Authority: WO
Inventors: Giammaria Giuliani; Raymond Rodriguez; Somen Nandi
Original assignee: Dermachip Inc.
Priority date: 2011-05-03
Filing date: 2012-05-03
Publication date: 2012-11-08
Also published as: EP2704728A1; US20140163118A1; EP2704728A4

Abstract

Described herein are expression signatures of genes and coordinately expressed gene networks associated with skin aging, methods used to determine the physiological age of skin, and methods used to screen for compounds used to reduce the visible signs of aging of the skin.

Description

IN THE PATENT COOPERATION TREATY

INTERNATIONAL PATENT APPLICATION

Title: Expression signatures of genes and gene networks associated with skin aging

Inventors: Giammaria Giuliani, Raymond Rodriguez, and Somen Nandi

Government sponsorship: None

Assignee: DERMACHIP INC. 719 2nd Street Suite 10, Davis, CA 95616, The USA

Relation to other applications: This application claims the benefit of and priority to US Provisional application No. 61/482,071 filed 03-MAY-2011.

[001 ] Field of the invention

[002] The invention relates to newly discovered expression signatures of coordinately expressed genes and gene networks associated with the physiological and pathological processes of skin aging and to methods for identifying and using compounds that regulate and in some cases recalibrate gene sets and gene pathways associated with skin ageing wherein such regulation and recalibration effectively reverses the effects of ageing of the skin to produce more youthful looking skin so that the skin is more supple, smooth and hydrated and has less wrinkles and fine lines typically associated with ageing.

[003] Background

[004] Skin aging is natural process associated with a number of pathophysiologies that can reduce quality of life and longevity. The goal of this study is to gain new insights into how skin ages as a function of time. The working hypothesis for this study is that skin aging results from the dysregulation of key regulatory genes (i.e., gene signatures) that control thousands of genes in multiple, interacting and overlapping networks and pathways. Dysregulation of gene functions leads to physiological aging of tissues, cells and subcellular components. This dysregulation may be the result of damage to the products of key regulatory genes (i.e., oxidative damage to regulatory proteins) or to transcriptional silencing of these genes by mutagenic and/or epigenetic processes. This study is the first of its kind to examine, in a comprehensive fashion, the gene expression profiles in human skin (specifically adult females who self -identified themselves as white) of various age and to identify gene networks and gene signatures associated with the aging process. This information should yield a small number of robust and predictive genes/gene-network whose expression can be used to: (a) understand the relationship between chronological age and physiological age and (b) assess the ability of compounds (e.g., drugs, natural products and dietary factors) to regulate and/or dysregulate genes and gene networks. The present study provides information that will guide future research and suggest new therapies for preventing, mitigating or treating skin pathologies related to aging.

[005] The skin is the largest human organ comprising about one sixth of total body weight. The skin performs a complex role in human physiology: serves as a barrier to the environment, and the sebum produced by some of its glands (sebaceous) have anti-infective properties. The skin acts as a channel for communication to the outside world, protects us from water loss, friction wounds, and impact wounds and uses specialized pigment cells to protect us from ultraviolet rays of the sun. Skin produces vitamin D in the epidermal layer, when it is exposed to the sun's rays. The skin helps regulate body temperature through sweat glands and helps regulate metabolism. The skin consists of three functional layers: Epidermis, the Dermis (or corium) and the Subcutis (or hypodermis).

[006] Various cell types are present in the skin. Keratinocytes is the most abundant cell type in the epidermis. These cells produce keratin proteins. Fibroblasts differentiate into cells that form the dermis and produce collagen and elastin. Melanocytes produce the pigment melanin that accumulates around the nuclei of the keratinocytes absorbing harmful ultraviolet (UV) light. Langerhans cells (macrophages) reside in the dermis mediating humoral and cellular immune functions. Merkel's cells, which are present in small numbers but are more numerous in the skin of the palms and soles of the feet, are sensory mechanical receptors that respond to certain stimuli such as pressure or touch. The epidermis is the outermost skin layer. As skin cells migrate to the surface, farther away from their source of nourishment, they flatten and shrink. They lose their nuclei, move out of the basal layer to the horny layer (stratum corneum), and die. This process, called keratinization, takes about 4 weeks. About 10 percent of epidermal cells are melanocytes that pigment the skin. The epidermis is differentiated into five layers: horny layer (stratum corneum); clear layer (stratum lucidum); granular layer (stratum granulosum); prickle-cell layer (stratum spinosum); and the basal layer (stratum basale). The dermis is the layer just below the outer keratinized epidermal layer. The dermis contains cells, water, collagen fibers, glycosaminoglycans and fibronectins that form a hydrated gel and are responsible for the high elasticity and tensile strength of the dermis. Embedded in this layer are lymph channels, blood vessels, nerve fibers, muscle cells, hair follicles, sebaceous glands, and sweat glands.

[007] Glycosaminoglycans are mucopolysaccharides present in the dermis that can bind large amounts of water. As the skin ages, the interweaving of the collagen fibers increases and the water-binding capacity diminishes and the skin tends to wrinkle. Glycosaminoglycans bind with the proteins in the connective tissue matrix to form proteoglycans. These proteoglycans form a gel-like material that can absorb and expel water like a sponge.

Glycosaminoglycans are subject to a continuous turnover. In contrast, the collagen fibers are only renewed when necessary, such as when injury is sustained. The ability of the skin to store water and thereby remain soft and supple depends in part on the presence of lipids, arginine, and other "natural moisturizing factors" (NMF) that originate from the cornification (differentiation) of the keratinocytes, for example, pyrrolidine carboxylic acid, and secretions from the sweat and sebaceous glands including urea, salts, and organic acids.

[008] The dermis also contains collagens. Type I collagen is the most abundant protein in skin connective tissue, which also contains other types of collagen (III, V, VII), elastin, proteoglycans, fibronectin, and other extracellular matrix proteins. Newly synthesized type I procollagen is secreted into the dermal extracellular space where it undergoes enzymatic -processing, arranging itself into a triple helix configuration. The triple helix complexes associate with other extracellular matrix proteins such as leucine-rich small proteoglycans, to form regularly arranged fibrillar structures. This process, called fibrillogenesis, results in formation of collagen bundles that are responsible for the strength and resiliency of the skin.

[009] Skin aging is influenced by several factors, including genetics, environmental exposure (ultraviolet (UV) irradiation, xenobiotics, and mechanical stress), hormonal changes, and metabolic processes (generation of reactive chemical compounds such as activated oxygen species, sugars, and aldehydes). Taken together, these factors lead to cumulative alterations of skin structure, function, and appearance. The influence of the environment, especially solar UV irradiation, is of considerable importance for skin aging. Skin aging due to UV exposure (photoaging) is superimposed on chronological skin aging. Historically, scientists considered photoaging and chronological skin aging as two distinct entities. Although the typical appearance of photoaged and chronologically aged human skin can be readily distinguished, recent evidence indicates that chronologically aged and UV-irradiated skin share important molecular features including altered signal transduction pathways that promote matrix-metalloproteinase (MMP) expression, decreased procollagen synthesis, and connective tissue damage. This concordance of molecular mechanisms suggests that UV irradiation accelerates many key aspects of the chronological aging process in human skin. Based on this relationship between UV irradiation and chronological aging, acute UV irradiation of human skin may serve as a useful model to study molecular mechanism of skin chronological aging.

[0010] At the tissue level, chronologically aged skin shows general atrophy of the extracellular matrix reflected by decreased number of fibroblasts, and reduced levels of collagen and elastin. The organization of collagen fibrils and elastin fibers is also impaired. This impairment is thought to result from both decreased protein synthesis that particularly affects types I and III collagens in the dermis and increased breakdown of extracellular matrix proteins.

[0011] Photodamaged skin is associated with increased epidermal thickness and alterations of connective tissue organization. The hallmark of photoaged skin is accumulation of amorphous elastin-containing material that resides beneath the epidermal dermal junction. Impairment of the fibrillar organization of collagen and elastin is typically more severe in photoaged skin, compared to sun-protected chronologically aged skin. The severity of photoaging is proportional to accumulated sun exposure and inversely related to the degree of skin pigmentation. Individuals with fair skin are more susceptible to solar UV-induced skin damage than darker-skinned individuals.

[0012] At the cellular level, one of the earliest detectable responses of human skin cells to UV irradiation is activation of multiple cytokine and growth factor cell surface receptors, including epidermal growth factor receptor (EGF-R), tumor necrosis factor (TNF) alpha receptor, platelet activating factor (PAF) receptor, insulin receptor, interleukin (IL-1) receptor, and platelet -derived growth factor (PDGF) receptor.

[0013] Activation of cell surface cytokine and growth factor receptors results in recruitment in cytoplasm of adaptor proteins that mediate downstream signaling. Assembly of these signaling complexes results in activation of small GTP-binding protein family members which are key upstream regulators of the certain MAP kinases. The action of certain GTP-binding proteins results in an increased formation of superoxide anions. This increased production of ROS likely participates in amplification of the signal leading to the activation of the downstream enzyme complexes such as MAP kinase. ROS are necessary participants in multiple MAP kinase pathways.

[0014] Increased intracellular ceramide content may also contribute to activation of the MAP kinase pathways by UV irradiation. UV-induced ceramide generation seems to be dependent on increased ROS production, since ceramide and ROS levels rise in parallel, and UV-induced ceramide production is inhibited by the free radical scavenger Vitamin E. MAP kinase activation results in induction of transcription factor AP-1 that is a major effector of the MAP kinase pathways. AP-1 regulates expression of many genes involved in the regulation of cellular growth and differentiation. Transcription of several MMP (matrix-metalloproteinase) family members is strongly regulated by AP-1. Several MMPs are upregulated by AP-1. These include MMP-1 (interstitial collagenase or collagenase 1) which initiates degradation of types I and III fibrillar collagens, MMP-9 (gelatinase B), which further degrades collagen fragments generated by collagenases, and MMP-3 (stromelysin 1), which degrades type IV collagen of the basement membrane and activates pro-MMP-1. MMP induction is, in part, responsible for UV-induced damage to skin connective tissue. Together, MMP-1, MMP-3, and MMP-9 have the capacity to completely degrade mature fibrillar collagen in skin. Consistent with this, increased collagen breakdown has been demonstrated within 24 h after UV irradiation in human skin in vivo. Thus, UV irradiation of human skin causes extracellular matrix degradation via induction of transcription factor AP-1 and subsequent increased MMP production. [0015] In addition to causing collagen breakdown, UV irradiation impairs new type I collagen synthesis. UV irradiation has been shown to decrease collagen production and impair organization of collagen fibrils in skin in vivo. In addition, increased breakdown of extracellular matrix proteins is also observed in UV-irradiated fibroblasts in vitro and in human skin in vivo. Down-regulation of type I collagen is mediated in part by UV- induced AP-1, which negatively regulates transcription of both genes that encode for type I procollagen (COL1A1 and COL1A2).

[0016] UV-induced down-regulation of collagen synthesis also occurs via paracrine mechanisms involving transforming growth factor-beta (TGF-beta) and other cytokines. TGF-beta is a major profibrotic cytokine, which regulates multiple cellular functions including differentiation, proliferation, and induction of synthesis of extracellular matrix proteins. The biological effects of TGF- beta are diverse and strongly dependent on its expression pattern and cell type. In human skin, TGF- beta inhibits growth of epidermal keratinocytes and stimulates growth of dermal fibroblasts. Moreover, TGF-beta induces synthesis and secretion of the major extracellular matrix proteins collagen and elastin. TGF-beta also inhibits expression of certain specific enzymes involved in the breakdown of collagen, including MMP-1 and MMP-3. TGF-beta also has the ability to affect gene expression by epigenetic modification of DNA. Exogenous TGF-beta was shown to induce and maintain expression of Foxp3 in regulatory T cells by demethylating a highly conserved region of the Foxp3 gene called Treg-specific demethylation region (TSDR) [J.K. Polansky et al., 2008. Eur. J. Immunol. 38: 1654-1663]. Both aging and UV irradiation induce molecular alterations that create skin aging. A major feature of aged skin is the reduction of types I and III procollagen synthesis. This reduction results in skin thinning and increased fragility. Both types I and III procollagen mRNA and protein expression are reduced in aged skin.

[0017] In addition to impaired collagen synthesis, increased production of several MMP family members, including MMP-1, MMP-2 (gelatinase A), MMP-3, and MMP-9 occurs in chronologically aged skin. With the exception of MMP-2, these MMPs are regulated by AP-1 and induced by UV irradiation. Interestingly, AP-1 expression is increased in aged human skin in vivo and aged skin fibroblasts in vitro. Oxidative stress is thought to be of primary importance in driving the aging process. The free radical theory of aging, first proposed several decades ago, envisions that the molecular basis of aging derives from accumulation, over a lifetime, of oxidative damage to cells resulting from excess ROS, which are produced as a consequence of aerobic metabolism.

Although skin possesses extremely efficient anti-oxidant activities, it has been demonstrated that during aging, ROS levels rise and anti-oxidant defenses decline. ROS are necessary participants in multiple MAP kinase pathways. MAPK activation results in induction of AP-1, which in turn, upregulates expression of MMPs. This scenario provides a plausible mechanism for the observed increased collagen degradation in aged human skin. [0018] In spite of existing differences, many critical molecular features of aged and UV-irradiated human skin bear striking similarities. It could be stated that these similarities reflect the central role that oxidative stress plays in UV irradiation-induced responses and aging in human skin. Viewed in this light, it is not surprising that UV irradiation and aging evoke similar molecular responses, since both are responding to oxidative stress. Nor is it surprising that the consequences of UV irradiation and aging have similar damaging impact on skin connective tissue.

[0019] Short description of the invention

[0020] The invention relates to expression signatures of genes and to coordinately expressed gene networks associated with skin aging. Additionally the invention relates to methods used to determine the physiological age of skin, and methods used to screen for compounds used to reduce the visible signs of aging of the skin.

[0021] The inventors have used high throughput expression screening and SAGE (Serial Analysis of Gene Expression) analysis to compare sets of skin tissue from two cohorts of individuals of substantially different ages. The inventors have discovered a large number of previously unknown expression signatures that are related to age. Additionally they have discovered a large and complex network of coordinately expressed genes and pathways that are associated with the physiological and pathological process of skin aging. These expression signatures are used to (i) determine the physiological age of skin, (ii) screen for compounds and compositions that are used to reduce the visible signs of aging of the skin, particularly to the prevention and reduction of skin wrinkles and to the production and maintenance of youthful looking skin.

[0022] Additionally, using the methods of the invention, compounds may be identified by the methods of the invention which compounds affect the expression of various genes within the skin that are involved in chronological-induced and UV -induced skin damage. The invention relates to methods for recalibrating the expression of genes, genetic networks, and cellular pathways in the human skin, primarily in the dermis, that have changed as a result of the chronological aging process. The invention also relates to combinations of natural compounds that produce synergistic effects on the expression of genes relevant to the reversal of skin aging and skin cancer risk reduction.

[0023] Description of the figures and tables

[0024] Figure 1. A regulatory module responsible for a single gene signature in one aging-related network. White = up-regulated, grey = down-regulated). This network which created using only top 100 up & down regulated genes illustrates the complexity of the aging process. This network is connected to other networks involved in skin aging as well as to aging in other tissues. It is the gene signatures in different but connected and interacting networks that determine the aging process. It is the genetic regulatory module (e.g., IL-8, IFNG) that will serve as targets for drugs and natural compounds to reverse skin aging. It is believed that the loss of connections between networks reduces the ability of the cell to make coordinated responses to external stimuli and thus the cause of disease and aging.

[0025] Figure 2. Master regulators correlate with the differential gene expression signatures between old and young cohorts. The X axis represents age from older (on the left) to younger (on the right). For each regulator (acronym boxed on the left hand side) two rows are shown, one of up-regulated genes and one of downregulated genes. It is very clear from this figure that certain genes and gene stets are upregulated in older skin samples and certain genes and gene sets are upregulated in younger skin samples. Low expression level in old cohorts is at the left of the bottom row which shows expression levels gradually increased to the right. The regulator gene symbols are listed at left side of the graph inside boxes. Each regulator has two rows of up and down regulated genes that are positioned according to their gene expression levels in the data sets. The P-value of the correlations between the gene expression signature of overall datasets (bottom row) and individual regulation patterns are listed right below each gene symbol. The lower the P-value, the high possibility the regulator has to be responsible for the signature changes. STAT1 = a member of the Signal Transducers and Activators of Transcription family of transcription factors. CEBPD = a CCAAT/enhancer-binding protein delta is a protein that in humans is encoded by the CEBPD gene. The protein encoded by this intronless gene is a bZIP transcription factor which can bind as a homodimer to certain DNA regulatory regions. MAF = a transcription factor that in humans is encoded by the MAF gene. RXRA = Retinoid X receptor alpha (RXR-alpha), also known as NR2B1 (nuclear receptor subfamily 2, group B, member 1) is a nuclear receptor. NR1H3 = Liver X receptor alpha (LXR-alpha) is a nuclear receptor protein. TFE3 = Transcription factor E3. NFATC1 = Nuclear factor of activated T-cells, cytoplasmic 1. RBL2 = Retinoblastoma-like protein 2. SMAD3 = Mothers against decapentaplegic homolog 3. NR2F1 = Nuclear Receptor subfamily 2, group F, member 1 is member of nuclear hormone receptor family of steroid hormone receptors.

[0026] Table 1. This table lists genes of interest with names and symbol and HGNC ID. Table 1 is used to look up the identity of genes listed on Table 4, which shows the degree of up or down-regulation for these genes.

[0027] Table 2. This is a list of cellular pathways and other functional categories based on the up- and down- regulated genes in Tablel showing the degree of enrichment based on the -2000 up and down regulated genes. A conservative cut-off with FDR of <=0.1 has been used to identify important pathways. [0028] Table 3. This table shows regulatory genes. This table shows a list of gene signatures and regulatory factors controlling the expression of skin-aging-related genes in various pathways and/or gene networks. Note that the gene described in italics at the end of the table are not part of Table 1 (which only includes the top 2000 up and down regulated genes); these italicized genes are part of the gene network regulators.

[0029] Table 4. This table shows the 2000 genes showing the greatest degree of up- or down-regulation and differential expression between the two age cohorts. This table shows the degree of differential expression for the top 2000 up and down regulated genes selected out of over 24000 human genes. 'Fc' means Fold Change represented in log 2 scale (these are relative numbers in comparison to expression of other genes so no units are appropriate). The fold-changes in gene expression are all relative to the young subjects. Thus a positive (+) value mean up-regulated in older subject, down-regulated in younger subjects. Negative (-) values means down- regulated in older subjects, up-regulated in younger subjects. HGNC is the standard identification number designated by HUGO Gene Nomenclature Committee. This data was gathered from a total of 32 subjects including 15 samples from the older group (age 59 - 75 year), and 17 samples from younger group (age 19 - 21 years). All samples were collected from healthy volunteer white females from an area of the body unexposed to sun (the buttock cheeks). Rest of the procedure are as described herein.

[0030] Table 5. This table shows a list of the top 35 genes/regulators controlling the expression of skin-aging- related genes in various genetic networks. This set of genes is selected from the larger list disclosed herein with the intention of supporting specific claims. The italicized genes were discovered during gene network analysis and are not part of Table 1. The inventors explicitly are not restricting the scope of the invention to these 35 genes.

[0031] Table 6. This table is a reduced list of the top 23 gene signatures controlling the expression of skin-aging- related genes in genetic networks.

[0032] General Representations Concerning the Disclosure

[0033] In this specification where reference is made to particular features of the invention it is to be understood that the disclosure of the invention in this specification includes all appropriate combinations of such particular features. The embodiments disclosed in this specification are exemplary and do not limit the invention. As used in this specification, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. The term "comprises" and grammatical equivalents thereof are used in this specification to mean that, in addition to the features specifically identified, other features are optionally present. The term "at least" followed by a number is used herein to denote the start of a range beginning with that number. Where reference is made in this specification to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously.

[0034] This specification incorporates by reference all documents referred to herein and all documents filed concurrently with this specification or filed previously in connection with this application, including but not limited to such documents which are open to public inspection with this specification. Additionally this application incorporates by reference US provisional application No. 61,198,235 filed Nov 3^rd 2008, and also to PCT/US09/063137, international filing date Nov 3^rd 2009, and also to the following patents and patent publications: US 6,569, 624 US 2004/0142335 US 2006/0275294 US 2003/0152947 US 2005/0089914 US 2007/0059711 US 2005/0250137 US 2005/0250137 US 2005/0053637 US 7,105,292 US 2007/0161022 US 6,692,916 US 2006/0134663 and US 2007/0148106. The specification also incorporates by reference the following: Anuurad, E., Yamasaki, M., Shachter, N., Pearson, TA. and Berglund, L. (2009). ApoE and ApoC-I polymorphisms: association of genotype with cardiovascular disease phenotype in African Americans. J Lipid Res 50(7): 1472-8. Deo, R. G, D. Reich, Tandon, A., Akylbekova, E., Patterson, N., Waliszewska, A.,

Kathiresan, S., Sarpong, D., Taylor, HA. Jr. and Wilson, J. G. (2009). Genetic differences between the determinants of lipid profile phenotypes in African and European Americans: the Jackson Heart Study. PLoS Genet 5(1): el000342. Van Dyke, A. L., M. L. Cote, Wenzlaff, AS., Land, S. and Schwartz, AG. (2009).

Cytokine SNPs: Comparison of allele frequencies by race and implications for future studies. Cytokine 46(2): 236-44.

[0035] Definitions

[0036] The term "signs of skin aging" refers to any anatomical visible indication that is generally associated with skin as a person gets older, including wrinkles, sagging, discoloration and reduced suppleness.

[0037] The term "recalibrating" when applied to the expression of genes, genetic networks, and cellular pathways refers to a change of adjustment of expression of one or more genes to produce a verisimilitude of a former state, such as the adjustment of expression of one or more genes listed in Table 1 so as to increase the production of glycosaminoglycans, proteoglycans, collagen etc.

[0038] The term " genetic network" or "genetic pathway" refers to two or more genes the expression of which is coordinated or related to a single physiological function such as the production of a particular protein or glycosaminoglycan.

[0039] The term "variant or derivative" when used in conjunction with a species such as a drug or other chemical entity is used to mean said drug or other chemical entity comprising at least one chemical modification, such as, but not limited to, a moiety, a radical group, a reactive group, a charged group, an uncharged group, an ion, or the like. The chemical modification can be either addition or removal of such moiety, group, ion, or the like. [0040] The term "drug" is used to mean any molecule that alters the physiology of an organism.

[0041] The term "protein" includes peptides.

[0042] The term "environmental stimulus" is used to mean any stimulus that in some way affects the physiology of an organism and that has its origins outside of the organism.

[0043] The term "a therapeutic amount" is used to mean an amount (of a substance) that produces a measurable effect related to the health of an organism.

[0044] The term "gene expression" is used to refer to the transcription of a gene or a part of a gene and is independent from translation. The expression of the gene or part thereof can be increased or it can be decreased. Translation of the expressed gene or part thereof can be increased or it can be decreased.

[0045] The term "gene regulatory network" (GRN) refers to a collection of genes or DNA segments in a cell which interact with each other directly or indirectly through their RNA and protein products. Genes may also interact with other constituents in the cell, thereby governing the rates at which genes in the network are transcribed into mRNA. Some proteins serve only to activate other genes, and these transcription factors that are the main players in regulatory networks or cascades. By binding to the promoter region at the start of other genes they turn them on, initiating the production of another protein, and so on. Some transcription factors are inhibitory.

[0046] The term "genetic regulatory module(s)" refers to a gene or group of genes whose action sets the transcription levels for other genes belonging to a network, which satisfy a certain arbitrary threshold criteria, such as 1.5 fold change with a significance p<0.01.

[0047] "HGNC" is the standard identification number designated by HUGO Gene Nomenclature Committee (http://www.genenames.org/). The HUGO Gene Nomenclature Committee is responsible for providing human gene naming guidelines and approving new, unique human gene names and symbols (short form abbreviations of full gene name). In addition to species-specific databases, approved gene names and symbols for human can be located in the National Center for Biotechnology Information's Entrez Gene database

(http://www.ncbi.nlm.nih. gov/sites/entrez?db=gene). See: Genetic nomenclature guide (1995). Trends Genet and The Trends In Genetics Nomenclature Guide (1998). Elsevier, Cambridge.

[0048] Detailed description of the invention

[0049] The invention relates to expression signatures of genes and to coordinately expressed gene networks associated with skin aging. Additionally the invention relates to methods used to determine the physiological age of skin, and methods used to screen for compounds used to reduce the visible signs of aging of the skin. [0050] The inventors have used high throughput expression screening and SAGE (Serial Analysis of Gene Expression) analysis to compare sets of skin tissue from two cohorts of individuals of substantially different ages. The inventors have discovered a large number of previously unknown expression signatures that are related to age. Additionally they have discovered a large and complex network of coordinately expressed genes and pathways that are associated with he physiological and pathological process of skin aging. The results have identified coordinated gene expression networks and coordination between metabolic pathways that were previously not known to be related to skin ageing. The results have identified a number of transcriptional activators and expression regulators that appear to play key roles in regulation of genes and pathways involved in skin ageing. For example, cholesterol biosynthesis and peroxisome lipid metabolism are both downregulated in the older population, but IL-6 synthesis is upregulated.

[0051] The newly discovered expression signatures may be used to (i) determine the physiological age of skin, (ii) screen for compounds and compositions that are used to reduce the visible signs of aging of the skin.

Information determined from SAGE analysis includes (1) a categorization of up- and down regulated genes in the younger and older subjects; (2) categorization of physiological and signal transduction pathways in which these genes are over-represented and (3) categorization of gene networks and genes that constitute hubs within the networks.

[0052] This disclosure further describes a method for reversing signs of skin aging and risk of skin cancer by recalibrating the expression of genes, genetic networks, and cellular pathways in the human skin, primarily in the dermis, that have changed as a result of the chronological aging process. Gene expression patterns, and the pathways they participate in, are restored to levels characteristic of a younger chronological age by treating the skin with specific combinations of natural compounds (e.g., phyto-chemicals, nutrients, minerals, vitamins, etc). Specific combinations of natural compounds are determined using informatic algorithms and high throughput screening. Natural compounds are delivered to the dermis topically with dermo-cosmetics and internally with oral supplements. Combinations of natural compounds are claimed that produce synergistic effects on the expression of genes relevant to the reversal of skin aging and skin cancer risk reduction. Natural compounds can affect gene expression directly (e.g., transcription factor agonists or antagonist) or indirectly (e.g., non-coding RNAs, epigenetic modifications, signalling receptor agonists or antagonist). The invention includes those natural compounds that produce synergistic effects on gene expression when administered both orally and topically. Also disclosed are those genes, gene networks, non-coding RNAs and epigenetic modifications associated with chronologically younger or older skin. [0053] The invention includes various specific embodiments such as, for example:

[0054] A method for delaying, ceasing or reversing signs of skin aging and risk of skin cancer by recalibrating the expression of genes, genetic networks, and cellular pathways in the human skin, primarily in the dermis, that have changed as a result of the chronological aging process wherein the genes are selected from the group consisting of specific genes are listed in Table 1, cellular pathways, and other functional categories are listed in Table 7, and gene signatures controlling the expression of aging-related genes in gene networks in Table 3 .

[0055] The method above wherein the genes recalibrated comprise one or more genes from Table 1 or genes selected from the group consisting of the genes of the functional categories listed in Table 7 and Table 3.

[0056] The method above wherein the genetic networks or cellular pathways recalibrated comprise one or more selected from the groups consisting of the genetic networks or cellular pathways listed in Table 7 and Table 3.

[0057] A method for reducing the signs of aging of the skin the method comprising applying to the skin a compound that was identified as having recalibrating potency with the method above.

[0058] A method for reducing the signs of aging of the skin the method comprising topically applying to the skin a compound "A" having anti-aging properties and further comprising orally administering a compound "B" having anti-aging properties.

[0059] The method above wherein compounds A and B, when administered contemporaneously, provide a synergistic effects on expression of genes of Table 1 or groups of Table 7 and Table 3.

[0060] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that alter the expression of genes involved in the biosynthesis or degradation of a substance selected from the 16 groups listed in Table 7.

[0061] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that increase the expression of genes involved in the biosynthesis of type I or type II collagen.

[0062] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that recalibrate the genes for enzymes listed in Table 1.

[0063] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that decrease production of MMPs.

[0064] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that reduce the rate of degradation of the extracellular matrix proteins in the dermis.

[0065] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the number of fibroblasts present in the dermis.

[0066] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the number of collagen fibrils or elastin fibers in the dermis.

[0067] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the number of collagen fibrils or elastin fibers in the dermis. [0068] A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the 3-dimensional extracellular matrix structure of collagen, elastin, and other extracellular matrix proteins in the dermis,

[0069] A composition for reducing the signs of aging of the skin the composition comprising combinations of natural compounds including phytochemicals, nutrients, minerals, vitamins, etc.

[0070] The invention also encompasses compositions of natural compounds, for external application to the skin, that reduce, delay, and/or reverse the signs of aging of the skin; composition of natural compounds, for internal application that reduce, delay, and/or reverse the signs of aging of the skin; compositions of natural compounds that produce synergistic effects on the expression of genes and/or gene products relevant to the reversal of skin aging and skin cancer risk reduction; compositions of natural compounds that affect and/or recalibrate the expression of various sets of genes, genetic networks, and/or cellular pathways in the human skin with the effect of reducing, delaying, and/or reversing the signs of aging of the skin; methods for reducing, delaying, and/or reversing the signs of aging of the skin by the external application and internal administration of claimed compounds; and methods for making above compounds and formulations; and methods for evaluating the efficacy of claimed compounds and formulations.

[0071] The present work also includes the identification of genetic regulatory modules responsible for gene signatures in aging-related networks. Fig. 1 shows a graphically displayed network (where white = up-regulated, grey = down-regulated genes) showing the complexity of the aging process. This network is connected to other networks involved in skin aging as well as to general aging in other tissues. Key to this and future work is the identification of nodes (genes) that connect networks together. It is the gene signatures in different but connected and interacting networks that determine the aging process. The genetic regulatory module (e.g. IL-6, IL-8, IFNG, CSF3) will serve as targets for drugs and natural compounds to reverse skin aging. The inventors believe that the lose of connections between networks reduces the ability of the cell to make coordinated responses to external stimuli and thus accelerates disease and aging.

[0072] The present work also includes the identification of genetic regulatory modules responsible for gene signatures in aging-related networks. Fig. 1 shows a graphically displayed network (where white = up-regulated, grey = down-regulated genes) showing the complexity of the aging process. This network is connected to other networks involved in skin aging as well as to general aging in other tissues. Key to this work is the identification of nodes (genes) that connect networks together. It is the gene signatures in different but connected and interacting networks that determine the aging process. The genetic regulatory module (e.g., IL-6, IL-8, IFNG, CSF3) provide targets for drugs and natural compounds to reverse skin aging. The inventors believe that the loss of connections between networks reduces the ability of the cell to make coordinated responses to external stimuli and thus accelerates disease and aging.

[0073] Methods of the invention

[0074] Gene expression analysis was performed on skin samples from a total of 32 subjects including 15 samples from the older group (age 59 - 75 year), and 17 samples from younger group (age 19 - 21 years). All samples were collected from healthy volunteer white females from an area of the body unexposed to sun (the white unexposed buttock cheeks).

[0075] The following steps were performed:

1) Obtaining of healthy skin from adult female who self-identified themselves as white of different ages.

2) Isolating mRNA from skin tissues for gene expression analysis using microarray technology.

3) Isolating DNA from skin tissues to assess DNA methylation status of key aging-related genes identified in specific aims 6.

4) Performing bioinformatics analysis of the gene expression dataset using various bio-computational and statistical tools.

5) Identification of genes, pathways and networks associated with the skin aging.

6) Identified a small number of genes whose expression can serve as predictors (i.e., biomarkers) of skin aging.

7) Correlation of chronological age with physiological age by use of biomarker genes/gene-net-works.

[0076] For data analysis the following steps were carried out.

[0077] (1) DNA Sequence fragments from the sequencing experiments were mapped to rna.fa files for Homo Sapiens from NCBI (ftp://ftp.ncbi.nih.gov/genomes/H_sapiens/PvNA/, rna.fa.gz, Size: 29793 KB, Last Modified: 11/22/10 7:40:00PM).

[0078] (2) BWA and SAMtools were used to map to sequences in rna.fa file (see item 1) and to count number of fragments match the same sequences with no more than two nucleotide differences. BWA algorithm was published at Li H. and Durbin R. (2009) Fast and accurate short read alignment with Burrows-Wtieeler Transform. Bioinformatics, 25:1754- 60. (incorporated by reference) and SAMtools was published at Li H.*, Handsaker B.*, Wysoker A., Fennell T., Ruan J., Homer N., Marth G., Abecasis G., Durbin R. and 1000 Genome Project Data Processing Subgroup (2009) The Sequence alignment/map (SAM) format and SAMtools.

Bioinformatics, 25, 2078-9. (incorporated by reference).

[0079] (3) R package DESeq 1.4.1 was used for statistics analysis. The DESeq algorithm was documented at Anders S. and Huber W. (2010) Differential expression analysis for sequence count data Genome Biology 11 :R106 (incorporated by reference). [0080] (4) Network analysis used STRING database. STRING database was published at von Mering C., Huynen M., Jaeggi D., Schmidt S., Bork P., Snel B., (2003), STRING: a database of predicted functional associations between proteins, Nucleic Acids Res. 2003 Jan 1 ;31(1):258-61 (incorporated by reference).

[0081] The dermis (inner most layer of the skin) and the epidermis (the outer most layer of the skin) were sampled together in this study. Volunteers, white women at least 18 years of age only, each provided a single, 4 mm deep biopsy of otherwise discarded skin tissue from the buttock.

[0082] Exclusion Criteria: Patients with the following criteria were excluded:

[0083] individuals with atopic dermatitis, psoriasis and other significant skin disorders

[0084] individuals with any serious medical condition

[0085] History of alcohol or drug abuse

[0086] Known hepatitis or HIV

[0087] Pregnant women

[0088] Significant allergic or adverse reaction to local anaesthetics

[0089] Blood clotting disorder

[0090] Faintness or vasovagal reaction with skin biopsies or procedures

[0091] Current smoker

[0092] The sex, race and health criteria were chosen to exclude as many variants as possible. A homogeneous population was chosen to reduce genetic variables.

[0093] Experimental Design

[0094] Identification of genes differentially expressed in healthy skin from subjects of different chronological age

[0095] Each 4mm buttock skin sample is immediately immersed into RNALater or snap frozen and stored at - 80°C until nucleic acid (RNA and DNA) extraction.

[0096] The samples undergo RNA and DNA extraction.

[0097] The extracted RNA is subjected to reverse transcription into cDNA, amplified, and processed for microarray analysis and also by Taqman qRT-PCR.

[0098] RNA sequenced are detected by using New Generation Sequencing (NGS), Illumna prpocedure, and data output and analysis is performed per standard analytical software. [0099] Average-linkage hierarchal clustering of data is applied using Gene Cluster software and results are displayed with Java TreeView. Analyses for differential expression include use of appropriate statistical limits for identified gene sets and both paired and multivariate statistical treatment. Gene and pathway identification is assisted by application of interaction gene network analyses for processes such as immune response, inflammation, wound repair, cell growth, apoptosis, cell migration, etc.

[00100] This method has lead to pathway-specific hypothesis testing and aids in identification of regulated genes and gene network modules. This treatment of the microarray data enables us to compare gene expression profiles in skin by age.

[00101] 2. DNA Methylation Status of Aging-Related Gene Signatures.

[00102] Based on the results of microarray data analysis, the DNA methylation status of candidate genes that are highly correlated with age (i.e., show the greatest change between young and old skin samples) were determined.

[00103] PureLink Genomic DNA Isolation kit (Invitrogen) was used to isolate DNA from half of the nucleic extraction samples.

[00104] This genomic DNA was subjected to bisulfite-specific PCR and sequencing of their 5' promoter regions to determine their DNA methylation status. The DNA is subjected to bisulfite conversion (EZ DNA-Methylation- Direct, Zymo Research).

[00105] Bisulfite-converted genomic DNA is subjected to bisulfite-specific PCR (BSP) using primers specific to the 5' end of the candidate genes. The BSP primers pairs that recognize methylated and unmethylated DNA are designed using Methyl-Primer Express vl.O (Lifetechnologies).

[00106] PCR is carried out in a 25 ul reaction volume using a Taq PCR Kit (New England BioLabs) and PCR products are resolved in 1.5% agarose gels. PCR bands are excised and purified using a Gel Extraction Kit (Qiagen) before ligating to pCR4-TOPO vector (Invitrogen), and transformed into DH5-alpha E. coli. Insert- containing plasmids are verified by PCR and EcoRI digestion before DNA sequencing.

[00107] Correlations between DNA hypermethylation in the 5 '-ends of aging-related gene signatures, gene expression levels and age are examined. DNA hypermethylation and low level expression of gene signatures in older skin samples are suggestive of epigenetic silencing of gene function, leading to aged skin.

[00108] 3. Skin Aging Gene Expression (SAGE) analysis

[00109] The initial experiment employed the following samples:

15 samples from the older group (age 59 - 75 years)

17 samples from younger group (age 19 - 21 years) 1 Sample has been rejected because of tissue inconsistency

1 Sample has been rejected because of the data quality

[00110] Transcript RNA from the samples was isolated and sequenced in 36bp x 36bp Paired End Reads. This gave about 45M raw reads per sample. About 94% passed quality control. About 84.5% mapped to human transcripts.

[00111] Results

[00112] The study has discovered a large number of genes and pathways that are differentially expressed between the old and young cohorts.

[00113] Additionally the study reveals that many genes and pathways display coordinated expression. This suggests that the coordinated pathways are subject to regulation by means of expression regulators such as transcription regulators, translation regulators, siRNAs and other suppressors of protein expression. Such regulators appear to have pleitropic effects on numerous genes not necessarily linked in series but found at disparate locations in the genome.

[00114] The results of the data analysis is shown Tables 1, 2, 3, 4, 5 and 6.

[00115] For example, cholesterol biosynthesis and peroxisome lipid metabolism are both downregulated in the older population, but IL-6 synthesis is upregulated.

[00116] Another pathway whose regulation is clearly affected by ageing is the T Cell Receptor (TCR) signaling pathway (see Table 7). T Cell Receptor (TCR) activation promotes a number of signaling cascades that ultimately determine cell fate through regulating cytokine production, cell survival, proliferation, and differentiation. An early event in TCR activation is phosphorylation of immunoreceptor tyrosine -base activation motifs (ITAMs) on the cytosolic side of the TCR/CD3 complex by lymphocyte protein- tyrosine kinase (Lck). The CD45 receptor tyrosine phosphatase modulates the phosphorylation and activation of Lck and other Src family tyrosine kinases, z-chain associated protein kinase (Zap-70) is recruited to the TCR/CD3 complex where it becomes activated, promoting recruitment and phosphorylation of downstream adaptor or scaffold proteins. Phosphorylation of SLP- 76 by Zap-70 promotes recruitment of Vav (a guanine nucleotide exchange factor), the adaptor proteins NCK and GADS, and an inducible T cell kinase (ITK). Phosphorylation of phospholipase C 1 (PLC_γl) by Itk results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the second messengers diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates PKCθ and the MAPK/Erk pathways, both promoting transcription factor NF-κΒ activation. IP3 triggers the release of Ca2+ from the ER, which promotes the entry of extracellular Ca2+ into cells through calcium release-activated Ca2+(CRAC) channels. Calcium-bound calmodulin (Ca2+/CaM) activates the phosphatase calcineurin, which promotes IL-2 gene transcription through the transcription factor NFAT. Feedback regulation at several points within these pathways allows for different outcomes, depending on the cell type and environment. The incorporation of signals from additional cell surface receptors (such as CD28 or LFA-1) further regulates cellular response.

Table 2. Pathway enriched based on the ~2000 up and down regulated genes. The presentation of enriched pathways based on DAVID database along with the source database where the pathways are from and have a enrichment calculation algorithm. We used a conservative cut-off with FDR <=0.1 to filter out important pathways. In case, you need a different cut-off, we have also attached the excel spread-sheet listing all the pathways pass the DAVID cut-off.

Claims

1. A method for reducing or reversing the visible signs of skin ageing, the method comprising up-regulating or down-regulating a specific pathway selected from the group consisting of the pathways listed in Table 2.

2. The method of claim 1 further comprising up-regulating or down-regulating the interaction between different but connected and interacting biosynthetic pathways listed in Table 2.

3. The method of claim 2 comprising up-regulating or down-regulating the interaction between two or more of the following regulators: STAT1, CEBPD, MAF, RXRA, NR2B1, NR1H3, TFE3, NFATC1, RBL, SMAD3, and NR2F1.

4. The method of claim 1 further comprising recalibrating the expression of two or more pathways expressed in human skin tissue the expression of which has changed as a result of aging wherein the pathways are selected form the IL-6 pathway, cholesterol biosynthesis, and proximal lipid metabolism pathway.

5. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that alter the expression of genes/pathways involved in the biosynthesis or degradation of IL-6 or cholesterol.

6. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that decrease or increase the expression of one or more genes selected from the genes listed Table 5.

7. A method for identifying a compound that will up-regulate or down-regulate the activity of one or more metabolic pathways associated with ageing, the method comprising: (i) providing a skin tissue sample, (ii) measuring the expression of a selected plurality of aging-related genes selected from the group consisting of the genes of Table 4, (iii) exposing skin tissue to a test compound, (iv) re-measuring the expression of the genes, (v) identifying a compound that changes the expression of the selected plurality of genes wherein the change in expression is at least two-fold.

8. The method of claim 7 wherein the plurality of aging-related genes comprise at least the genes encoding interleukin, colony stimulating factor 3, and interferon gamma.

9. The method of claim 7 wherein the plurality of aging-related genes comprise at least the genes encoding IL6, BARX1, CFS3, IFNG, CSAG3, PRHOXNB, IP8, LPA, CXCL2, FOXD4L6, OR1L8, IGLL5, and CXCL9.

10. The method of claim 7 wherein the plurality of aging-related genes comprise at least the genes encoding interleukin 6, colony stimulating factor 3, interferon gamma, interleukin 8, chemokine (C-X-C motif) ligand 2, chemokine (C-X-C motif) ligand 9, chemokine (C-X-C motif) ligand 10, and chemokine (C-X-C motif) ligand 1.

11. The method of claim 7 wherein the plurality of aging-related genes are shown in table 4 to be differentially expressed by a factor of at least fc (log2) of at least 4.

12. The method of claim 8 wherein the plurality of aging-related genes are shown in table 4 to be differentially expressed by a factor of at least fc (log2) of at least 4.

13. The method of claim 9 wherein the plurality of aging-related genes are shown in table 4 to be differentially expressed by a factor of at least fc (log2) of at least 4.

14. The method of claim 10 wherein the plurality of aging-related genes are shown in table 4 to be differentially expressed by a factor of at least fc (log2) of at least 4.

15. A method for identifying new compounds that alter aging-related gene expression profiles and signatures, the method comprising: (i) providing a skin tissue sample, (ii) measuring the expression of a selected plurality of aging-related genes selected from Table 1 , (iii) exposing skin tissue to a test compound, (iv) re -measuring the expression of the genes, (v) identifying a compound that changes the expression of the selected plurality of genes wherein the change in expression is at least two-fold.

16. A method for delaying, ceasing or reversing signs of skin aging and risk of skin cancer by recalibrating the expression of genes, genetic networks, and cellular pathways in the human skin, primarily in the dermis, that have changed as a result of the chronological aging process wherein the genes are selected from the group consisting of specific genes are listed in Table 1, cellular pathways, and other functional categories are listed in Table 2.

17. The method of claim 16 wherein the genes recalibrated comprise one or more genes from Table 1 or genes selected from the group consisting of the genes of the functional categories listed in Table 2 and Table 3.

18. The method of claim 16 wherein the genetic networks or cellular pathways recalibrated comprise one or more selected from the groups consisting of the genetic networks or cellular pathways listed in Table 2 and Table 3.

19. A method for reducing the signs of aging of the skin the method comprising applying to the skin a compound having recalibrating potency according to the method of claim 15.

20. A method for reducing the signs of aging of the skin the method comprising topically applying to the skin a compound "A" having anti-aging properties and further comprising orally administering a compound "B" having anti-aging properties.

21. The method of claim 20 wherein compounds A and B, when administered contemporaneously, provide a synergistic effects on expression of genes of Table 1 or groups of Table 2 and Table 3.

22. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that alter the expression of genes involved in the biosynthesis or degradation of a substance selected from the groups listed in Table 2.

23. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that increase the expression of genes involved in the biosynthesis of type I or type II collagen.

24. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that recalibrate the genes for enzymes listed in Table 1.

25. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that decrease production of MMPs.

26. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that reduce the rate of degradation of the extracellular matrix proteins in the dermis.

27. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the number of fibroblasts present in the dermis.

28. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the number of collagen fibrils or elastin fibers in the dermis.

29. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the number of collagen fibrils or elastin fibers in the dermis.

30. A composition for reducing the signs of aging of the skin, the composition comprising one or more substances that maintain or increase the 3-dimensional extracellular matrix structure of collagen, elastin, and other extracellular matrix proteins in the dermis,

31. A composition for reducing the signs of aging of the skin the composition comprising combinations of natural compounds including phytochemicals, nutrients, minerals, and vitamins.

32. A method for reversing skin ageing by targeting a genetic regulatory module selected from the group consisting of IL-6, IL-8, IFNG, and CSF3.

33. A method for delaying, ceasing or reversing signs of skin aging and risk of skin cancer by recalibrating the expression of genes, genetic networks, and cellular pathways in the human skin, primarily in the dermis, that have changed as a result of the chronological aging process wherein the genes are selected from the group consisting of specific genes are listed in Table 1, cellular pathways, and other functional categories are listed in Table 7, and a list of gene signatures controlling the expression of aging-related genes in various gene networks in Table 3, the method comprising applying to the skin one of more natural compounds selected from the group consisting of: Acerola, Vitamin C, Algae Extract, Alginate, Allantion, Aloe Vera, Apricot Seed Oil, Arbutin, Arnica Extract, Ascorbic Acid, Avocao Oil, Barley Extract, and Bearberry Extract.