A ging is a complex process dictated by an inherent genetic program and accelerated by environmental damage to genes and their protein products. Aging reduces maximal capacity and functional reserve of all organs in the body, ultimately to a point incompatible with life. This article reviews the recognized age-associated changes in skin and their pathophysiology and mechanism of treatment responsiveness, when known. It also explores current thoughts regarding the molecular basis of aging and its evolutionary value. Clinical Impact of Skin Aging Human skin is the body’s dynamic interface with the environment. As such, its roles include barrier function, chemical clearance, sensory perception, mechanical protection, wound healing, immune surveillance, thermoregulation, sebum production, vitamin D production and DNA repair.1 All of these functions decline with age, usually by 1/3 to 2/3 of young adult levels by the eighth decade. These losses predispose the elderly to a variety of minor and major health problems, both cutaneous and systemic, that include irritant contact dermatitis, intertrigo, skin cancer, chronic ulcers, heat stroke, hypothermia, osteomalacia and herpes zoster infection. In contrast to aging in most other body organs, however, skin aging impacts appearance and is of concern to individuals even when function is adequate to insure good health. The skin undergoes visible changes during life, which include those attributable to the passage of time alone (intrinsic aging) and superimposed changes due to the effects of chronic sun damage (extrinsic aging or photoaging) that are often far more pronounced and cosmetically disturbing. Intrinsic Aging of the Skin Clinically, aged skin is thin, pale, dry and wrinkled, with multiple benign neoplasms, such as seborrheic keratoses (see photo above).1,2 Hallmarks of aged skin, summarized in the table “Histologic Features of Aging Human Skin” on page 58, include epidermal and dermal atrophy with flattening of the dermal-epidermal junction (DEJ). This atrophy reflects a reduction in the proliferative keratinocyte population normally located in the rete ridges, in specialized epidermal cell populations, such as melanocytes and Langerhans cells, and loss of fibroblasts, blood vessels and appendages in the dermis. Ultimately, the changes at the DEJ impair cytokine-mediated communication as well as the transfer of nutrients and oxygen between dermis and epidermis.1 Overall, the dermis thins by 20% in the elderly,1 although thinning of intrinsically aged skin becomes most significant after the eighth decade.3 Collagen content per unit area of skin surface decreases by about 1% per year throughout adult life.4 Cultured skin fibroblasts have shown an age-associated increase in collagenase mRNA with a concomitant decrease in the rate of collagen synthesis.5 Dermal collagenase activity increases, leading to increased collagen degradation.6 Elastic fibers become fragmented with small cysts and lacunae, especially at the dermal-epidermal junction.7 There is a decrease in the overall vascularity of skin with age. Interestingly, in contrast to extrinsically aged skin, intrinsically aged skin has minimal pigmentary changes, although the density of melanocytes decreases progressively during adulthood by about 10% per decade.8 Extrinsic Aging of the Skin What accounts for the undesirable appearance of sun-damaged skin? A major contributing factor is the degradation of collagen and other components of the extracellular matrix (ECM). Type I collagen is a major component of dermal connective tissue, giving skin both strength and resiliency. Other components of the dermal matrix include type III collagen, elastin, proteoglycans and fibronectin. The best studied detrimental effects of sun-induced “premature skin aging” are destruction and disorganization of collagen. Increased release and activity of matrix metalloproteases (MMPs)9 and cytokines are induced by sun exposure, leading to increased collagen degradation. Cigarette smoking can compound the adverse effects of sun exposure, giving rise to coarse wrinkling and an increased risk of skin cancer.10-12 Additional features of sun-exposed skin include telangiectasias, prominence of pores, pre-cancerous lesions and a characteristic “bronzed” or chronically hyperpigmented appearance,1,2 associated with an increased density of melanocytes.8 The clinical and histologic characteristics of photoaged skin are summarized in “Features of Actinically Damaged Skin” on page 58. In contrast to sun-protected sites, the epidermis of sun-damaged skin may be either severely atrophic or hyperplastic.2 There are keratinocyte nuclear atypia and disturbed epidermal maturation,2 presenting clinically as actinic keratoses (AKs). Additionally, sun-damaged skin shows increased cellularity, prominent elastosis and decreased vascularity.13 A histologic hallmark of actinically damaged skin is elastosis,14,15 deposition of amorphous dermal elastin and collagen. In sun-protected skin, the amount of elastin increases slightly and also undergoes structural alterations,13,16,17 although these changes are subtle compared to those in actinically damaged skin. The clinical manifestations of photoaging depend on skin type and history of sun exposure. Skin types I and II tend to display freckling, likely reflecting mutational events, and eventually develop pre-cancerous lesions, such as AKs and non-melanoma and melanoma skin cancer. Phototype I or II skin also manifests proliferative exhaustion in the form of epidermal atrophy, focal depigmentation and pseudoscars (see photo on below). In constrast, skin types III and IV initially tan and later develop lentigines, epidermal thickening and other hypertrophic responses. In summary, patients with skin types I and II primarily undergo cell loss, mutation and dysplasia; while skin type III and IV patients tend to display protective hyperplasia. Retinoid Therapy Treatment of aging skin customarily involves the utilization of various cosmeceuticals, including hydroxy acids, growth factors, antioxidants, vitamins, hormones and retinoids.18 The scientific basis for the use of each class of skin products varies from very strong to non-existent. This section will review the best studied and only FDA-approved agent for the management of skin aging/photoaging: tretinoin (all trans-retinoic acid, at-RA), the active metabolite of vitamin A known to regulate embryogenesis and post-natal epithelial homeostasis at least in part by binding its nuclear receptors and modifying gene transcription.19 Tretinoin is a prescription drug sold under several brand names and generically. Retinol, which cells can metabolize to at-RA, is present in many cosmeceuticals, but controlled studies of its efficacy for aging/photoaging are not available. Early studies demonstrating the clinical and histologic efficacy of topical tretinoin for the treatment of photoaged skin were conducted in UV-irradiated hairless mice.20 In addition to the clinical amelioration of wrinkles in the mice, dermal changes included a “reconstruction zone”13 of new, structurally normal collagen in the papillary dermis.20 This zone of new collagen was later also demonstrated in human facial skin following topical tretinoin application.13 As described by the University of Michigan group who have performed many of the human studies, epidermal changes observed following application of tretinoin 0.05% to 0.1% to photoaged skin include increased cellular proliferation, a thickened granular layer, increased number of anchoring fibrils, and more normal appearing collagen in the papillary dermis.21 Rafal et al.,22 conducted a randomized, double-blinded study over a 10-month period on the efficacy of 0.1% tretinoin for the treatment of solar lentigines, another clinical manifestation of photodamage. The overall clinical response was evaluated by a single investigator in the majority of cases using degrees of color change. Clinical lightening was noted in 83% of treated facial lesions compared to 29% of controls. Pre-treatment and post-treatment histologic specimens were examined by a blinded observer using a semi-quantitative scale for various characteristics including epidermal pigmentation, spongiosis, stratum corneum compaction and dermal inflammation. Epidermal thickness was measured directly. The degree of epidermal pigmentation in the tretinoin group decreased by 35%, while it increased by 34% in the vehicle-treated group. To determine if topical application of tretinoin could reverse or repair effects of photodamage, particularly the degradation of collagen, Griffiths et al., used a mouse monoclonal IgG1 antibody that recognizes the aminopropeptide portion of pro-collagen I to provide an indirect measure of collagen I formation.23 Fifteen patients with photodamaged skin were treated for 10 to 12 months with tretinoin 0.1% daily, and biopsy specimens at baseline and at the conclusion of the study were compared and stained with the anti-body. Collagen I synthesis, which is reduced in photoaged skin, was partially restored with an increase of 80% over baseline values after tretinoin treatment, while vehicle control users experienced a 14% decrease. The finding of reduced levels of type I pro-collagen (indirect measure of collagen I) in photoaged skin was later verified along with reduced levels of type III pro-collagen.24 Matrix metalloproteases (MMPs) are proteolytic enzymes known to degrade collagen and components of the dermal ground substance. In a series of elegant experiments, Fisher et al., demonstrated that low-dose UVB irradiation of human skin in vivo induces matrix-degrading MMPs (collagenase, gelatinase, stromelysin) in a dose-dependent fashion.9 The genes encoding these inducible collagen and ECM degrading enzymes were up-regulated by the transcription factors AP-1 and NF-kB. In addition, AP-1 blocked collagen gene expression in dermal fibroblasts, impairing collagen synthesis. NF-kB amplified AP-1 and NF-kB release, resulting in further photodamage. Pre-treatment of human skin with topical tretinoin substantially blocked UVB induction of the transcription factor AP-1 binding to DNA and upregulation of mRNA, protein levels and activity for gelatinase and collagenase.9 The authors concluded that a major mechanism by which topical tretinoin repairs and prevents further photoaging is blocking induction of MMPs via AP-1. MAP (mitogen activated protein) kinase pathways transduce signals from growth factors, cytokines and other effectors of UVB damage. MAP kinase activation by low-dose UVB induces c-Jun, a component of transcription factor AP-1. Fisher et al.,25 showed that topical pre-treatment of human skin with tretinoin inhibits UV induction of c-Jun in human skin and consequently of AP-1 with blunting of MMP gene transcription. Ultimately, this prevents collagen degradation. The Michigan group also investigated the effect of UV irradiation on the regulation of pro-collagen synthesis in human skin in vivo, specifically the role of c-Jun, and asked if these effects could be reversed with tretinoin 0.1%.26 After a single UV exposure equivalent to 2 MEDs (minimal erythema doses), a significant reduction in type I and type III pro-collagen mRNA and protein was found. Multiple UV exposures of 1 to 4 MEDs prolonged the suppression of collagen synthesis. They then investigated the role of c-Jun in UV inhibition of type I pro-collagen gene expression in primary adult human skin fibroblasts. The fibroblasts were transfected with a CAT reporter gene under the control of type I (alpha-2) pro-collagen promoter, which contains the binding site for AP-1 (composed of Jun and Fos proteins), a negative regulator of pro-collagen transcription. UV irradiation rapidly induced c-Jun and reduced promoter activity. Lastly, the group pre-treated patients with 0.1% tretinoin and demonstrated preservation of type I and type III pro-collagen mRNA and protein levels, versus the reductions observed in UV-irradiated, vehicle treated skin. In summary, the above research has yielded several important lessons: • UV irradiation inhibits pro-collagen synthesis in human skin in vivo • Collagen synthesis is substantially reduced in chronically photoaged human skin in vivo • Tretinoin treatment of photoaged skin increases collagen synthesis • UV irradiation upregulates enzymes that degrade collagen in human skin in vivo, via induction of c-Jun, a component of the AP-1 transcription factor • Pre-treatment of skin with tretinoin inhibits UV induction of c-Jun and protects skin against loss of collagen. Telomeres and Aging Skin Why do we age? Let’s consider the possibility that aging represents an unintended and, until recently, largely irrelevant consequence of a highly effective cancer-prevention mechanism that has evolved over the millennia in multicellular higher organisms. This phenomenon centrally involves telomeres. It is a form of “antagonistic pleiotropy”27 that trades the protective effects of limited cellular proliferative capacity (no cancer) against its eventual detrimental effects, such as organ atrophy and poor wound healing. In a primitive world where humans rarely lived to age 40, both cancer and age-associated diseases were rare. As civilization outpaced evolution, however, “successful” members of society, those who lived for more than 40 years, began to experience problems due to failure of the mechanism (cancer) and simply to its existence (aging). Telomeres are tandem repeats of the DNA base sequence thymine-thymine-adenine-guanine-guanine-guanine (TTAGGG) and its paired complementary sequence at the end of each mammalian chromosome, consisting of 10,000 or so base pairs in humans. Because of the end replication problem, the inability of DNA polymerase to copy the final bases on each chromosome, with each round of cell division the telomere shortens. After a finite number of cell divisions (approximately 60 in human cells), the telomeres become “critically short” and the cell will no longer divide, regardless of the stimulation. In this way, telomeres function as the “biologic clock” and cause cells to enter a state termed replicative senescence.28 Structurally, the telomere is double stranded, except for a 3' guanine-rich single-stranded overhang that extends approximately 75 to 300 bases, repeats of TTAGGG. This overhang is normally concealed in a protective loop that is stabilized by binding proteins, the most important being telomeric repeat binding factor 2 (TRF2).29,30 If TRF2 is sequestered by a dominant negative construct, the telomere linearizes and the 3' overhang is digested, activating ATM (ataxia telangiectasia mutated protein, important for normal cell division and coordinating DNA-damage responses) and subsequently p53 (see diagram above). This leads to apoptosis in lymphocytes31 and senescence in human fibroblasts,30 known responses to acute DNA damage that also have been documented to occur with critical telomere shortening. Thus, experimental telomere loop disruption with TRF 2DN, replicative senescence after multiple rounds of cell division and acute DNA damage all cause identical responses: apoptosis in some cell types (such as lymphocytes) and permanent growth arrest in others (such as fibroblasts). Moreover, extensive studies have shown the apoptotic and senescent responses are mediated by identical proteins. Our group has shown that exogenous treatment of lymphocytes or fibroblasts with DNA oligonucleotides homologous to the telomere 3' overhang (T-oligos) mimics the effects of telomere disruption, causing these cells to undergo apoptosis or senescence, depending on cell type, mediated by ATM and the tumor suppressor protein p53.32 Treatment with T-oligos can trigger these and other DNA-damage like responses, including increased melanogenesis,33-35 cell cycle arrest35,36 and enhanced DNA repair capacity,37,38 in the absence of true DNA damage or telomere disruption. In combination, these data suggest that the 3' telomeric overhang sequence may be exposed by telomere shortening during chronologic aging or by acute DNA damage and that this event is mimicked by the exogenous treatment of cells with T-oligos. We hypothesize that exposure of the TTAGGG sequence activates a signaling pathway leading through ATM and p53 to transient growth arrest, apoptosis, adaptive differentiation or senescence (see diagram below). It further appears that the exact consequence of activating this pathway depends on cell type, intensity and duration of the initial signal. Skin experiences both intrinsic aging and chronic photodamage. The acknowledged overlap and mutual reinforcement of these processes, leading to “premature aging” or “photoaging,” might logically result from shared pathophysiologic mechanisms. The known and postulated roles of telomeres offer an attractive possible explanation for these observations (see diagram at left). Over many decades, epidermal stem cells, melanocytes and fibroblasts undergo multiple rounds of cell division with associated telomere shortening. In the event of UV irradiation or other environmental damage, additional compensatory cell division is likely. Simultaneously, cells are exposed to low-grade oxidative stress from normal cellular metabolism, stress again compounded by UV irradiation or other environmental exposures, leading to DNA damage, preferentially in the form of 8-oxo-guanine. UV irradiation further damages DNA through production of photoproducts, primarily cyclobutane pyrimidine dimers. Through telomere disruption and telomere-based DNA damage responses, these photo-products may upregulate DNA repair capacity and induce tanning or they may cause mutations in coding DNA that lead to cancer (see diagram below). Through telomere signaling, cells can also either senesce or undergo apoptosis, both protective anti-cancer responses. The proficiency and nature of these protective responses determines the character of photoaging. Adequate responses (possibly represented by skin types III/IV) allow people to tan well and to incur fewer mutations, whereas inadequate responses (possibly represented by skin types I/II) favor cell death or senescence, poor tanning, a high mutation rate and, ultimately, cancer. The ability of T-oligos to trigger such protective effects as enhanced melanogenesis (tanning), enhanced DNA repair capacity, reduced mutagenesis and enhanced antioxidant defenses, in the absence of DNA damage, suggests that unwanted aging/photoaging changes may someday be avoidable without disabling nature’s clever cancer prevention mechanism. Conclusion Much is now known about aging and photoaging at the physiologic and morphologic levels. Break-through studies over the past 15 years have partially elucidated the mechanisms by which one innate regulator of skin development and maintenance, tretinoin or at-RA influences these processes. The work has also provided our patients with a first generation of truly effective anti-aging therapies for their skin. Very recent research into the role of telomeres in regulating aging and responses to environmental DNA damage now provides a big picture in which to understand both intrinsic aging and photoaging. It is tantalizing to imagine that these new insights may also result in products to prevent and treat the unwanted consequences of skin aging.
Skin Aging and Photoaging
A ging is a complex process dictated by an inherent genetic program and accelerated by environmental damage to genes and their protein products. Aging reduces maximal capacity and functional reserve of all organs in the body, ultimately to a point incompatible with life. This article reviews the recognized age-associated changes in skin and their pathophysiology and mechanism of treatment responsiveness, when known. It also explores current thoughts regarding the molecular basis of aging and its evolutionary value. Clinical Impact of Skin Aging Human skin is the body’s dynamic interface with the environment. As such, its roles include barrier function, chemical clearance, sensory perception, mechanical protection, wound healing, immune surveillance, thermoregulation, sebum production, vitamin D production and DNA repair.1 All of these functions decline with age, usually by 1/3 to 2/3 of young adult levels by the eighth decade. These losses predispose the elderly to a variety of minor and major health problems, both cutaneous and systemic, that include irritant contact dermatitis, intertrigo, skin cancer, chronic ulcers, heat stroke, hypothermia, osteomalacia and herpes zoster infection. In contrast to aging in most other body organs, however, skin aging impacts appearance and is of concern to individuals even when function is adequate to insure good health. The skin undergoes visible changes during life, which include those attributable to the passage of time alone (intrinsic aging) and superimposed changes due to the effects of chronic sun damage (extrinsic aging or photoaging) that are often far more pronounced and cosmetically disturbing. Intrinsic Aging of the Skin Clinically, aged skin is thin, pale, dry and wrinkled, with multiple benign neoplasms, such as seborrheic keratoses (see photo above).1,2 Hallmarks of aged skin, summarized in the table “Histologic Features of Aging Human Skin” on page 58, include epidermal and dermal atrophy with flattening of the dermal-epidermal junction (DEJ). This atrophy reflects a reduction in the proliferative keratinocyte population normally located in the rete ridges, in specialized epidermal cell populations, such as melanocytes and Langerhans cells, and loss of fibroblasts, blood vessels and appendages in the dermis. Ultimately, the changes at the DEJ impair cytokine-mediated communication as well as the transfer of nutrients and oxygen between dermis and epidermis.1 Overall, the dermis thins by 20% in the elderly,1 although thinning of intrinsically aged skin becomes most significant after the eighth decade.3 Collagen content per unit area of skin surface decreases by about 1% per year throughout adult life.4 Cultured skin fibroblasts have shown an age-associated increase in collagenase mRNA with a concomitant decrease in the rate of collagen synthesis.5 Dermal collagenase activity increases, leading to increased collagen degradation.6 Elastic fibers become fragmented with small cysts and lacunae, especially at the dermal-epidermal junction.7 There is a decrease in the overall vascularity of skin with age. Interestingly, in contrast to extrinsically aged skin, intrinsically aged skin has minimal pigmentary changes, although the density of melanocytes decreases progressively during adulthood by about 10% per decade.8 Extrinsic Aging of the Skin What accounts for the undesirable appearance of sun-damaged skin? A major contributing factor is the degradation of collagen and other components of the extracellular matrix (ECM). Type I collagen is a major component of dermal connective tissue, giving skin both strength and resiliency. Other components of the dermal matrix include type III collagen, elastin, proteoglycans and fibronectin. The best studied detrimental effects of sun-induced “premature skin aging” are destruction and disorganization of collagen. Increased release and activity of matrix metalloproteases (MMPs)9 and cytokines are induced by sun exposure, leading to increased collagen degradation. Cigarette smoking can compound the adverse effects of sun exposure, giving rise to coarse wrinkling and an increased risk of skin cancer.10-12 Additional features of sun-exposed skin include telangiectasias, prominence of pores, pre-cancerous lesions and a characteristic “bronzed” or chronically hyperpigmented appearance,1,2 associated with an increased density of melanocytes.8 The clinical and histologic characteristics of photoaged skin are summarized in “Features of Actinically Damaged Skin” on page 58. In contrast to sun-protected sites, the epidermis of sun-damaged skin may be either severely atrophic or hyperplastic.2 There are keratinocyte nuclear atypia and disturbed epidermal maturation,2 presenting clinically as actinic keratoses (AKs). Additionally, sun-damaged skin shows increased cellularity, prominent elastosis and decreased vascularity.13 A histologic hallmark of actinically damaged skin is elastosis,14,15 deposition of amorphous dermal elastin and collagen. In sun-protected skin, the amount of elastin increases slightly and also undergoes structural alterations,13,16,17 although these changes are subtle compared to those in actinically damaged skin. The clinical manifestations of photoaging depend on skin type and history of sun exposure. Skin types I and II tend to display freckling, likely reflecting mutational events, and eventually develop pre-cancerous lesions, such as AKs and non-melanoma and melanoma skin cancer. Phototype I or II skin also manifests proliferative exhaustion in the form of epidermal atrophy, focal depigmentation and pseudoscars (see photo on below). In constrast, skin types III and IV initially tan and later develop lentigines, epidermal thickening and other hypertrophic responses. In summary, patients with skin types I and II primarily undergo cell loss, mutation and dysplasia; while skin type III and IV patients tend to display protective hyperplasia. Retinoid Therapy Treatment of aging skin customarily involves the utilization of various cosmeceuticals, including hydroxy acids, growth factors, antioxidants, vitamins, hormones and retinoids.18 The scientific basis for the use of each class of skin products varies from very strong to non-existent. This section will review the best studied and only FDA-approved agent for the management of skin aging/photoaging: tretinoin (all trans-retinoic acid, at-RA), the active metabolite of vitamin A known to regulate embryogenesis and post-natal epithelial homeostasis at least in part by binding its nuclear receptors and modifying gene transcription.19 Tretinoin is a prescription drug sold under several brand names and generically. Retinol, which cells can metabolize to at-RA, is present in many cosmeceuticals, but controlled studies of its efficacy for aging/photoaging are not available. Early studies demonstrating the clinical and histologic efficacy of topical tretinoin for the treatment of photoaged skin were conducted in UV-irradiated hairless mice.20 In addition to the clinical amelioration of wrinkles in the mice, dermal changes included a “reconstruction zone”13 of new, structurally normal collagen in the papillary dermis.20 This zone of new collagen was later also demonstrated in human facial skin following topical tretinoin application.13 As described by the University of Michigan group who have performed many of the human studies, epidermal changes observed following application of tretinoin 0.05% to 0.1% to photoaged skin include increased cellular proliferation, a thickened granular layer, increased number of anchoring fibrils, and more normal appearing collagen in the papillary dermis.21 Rafal et al.,22 conducted a randomized, double-blinded study over a 10-month period on the efficacy of 0.1% tretinoin for the treatment of solar lentigines, another clinical manifestation of photodamage. The overall clinical response was evaluated by a single investigator in the majority of cases using degrees of color change. Clinical lightening was noted in 83% of treated facial lesions compared to 29% of controls. Pre-treatment and post-treatment histologic specimens were examined by a blinded observer using a semi-quantitative scale for various characteristics including epidermal pigmentation, spongiosis, stratum corneum compaction and dermal inflammation. Epidermal thickness was measured directly. The degree of epidermal pigmentation in the tretinoin group decreased by 35%, while it increased by 34% in the vehicle-treated group. To determine if topical application of tretinoin could reverse or repair effects of photodamage, particularly the degradation of collagen, Griffiths et al., used a mouse monoclonal IgG1 antibody that recognizes the aminopropeptide portion of pro-collagen I to provide an indirect measure of collagen I formation.23 Fifteen patients with photodamaged skin were treated for 10 to 12 months with tretinoin 0.1% daily, and biopsy specimens at baseline and at the conclusion of the study were compared and stained with the anti-body. Collagen I synthesis, which is reduced in photoaged skin, was partially restored with an increase of 80% over baseline values after tretinoin treatment, while vehicle control users experienced a 14% decrease. The finding of reduced levels of type I pro-collagen (indirect measure of collagen I) in photoaged skin was later verified along with reduced levels of type III pro-collagen.24 Matrix metalloproteases (MMPs) are proteolytic enzymes known to degrade collagen and components of the dermal ground substance. In a series of elegant experiments, Fisher et al., demonstrated that low-dose UVB irradiation of human skin in vivo induces matrix-degrading MMPs (collagenase, gelatinase, stromelysin) in a dose-dependent fashion.9 The genes encoding these inducible collagen and ECM degrading enzymes were up-regulated by the transcription factors AP-1 and NF-kB. In addition, AP-1 blocked collagen gene expression in dermal fibroblasts, impairing collagen synthesis. NF-kB amplified AP-1 and NF-kB release, resulting in further photodamage. Pre-treatment of human skin with topical tretinoin substantially blocked UVB induction of the transcription factor AP-1 binding to DNA and upregulation of mRNA, protein levels and activity for gelatinase and collagenase.9 The authors concluded that a major mechanism by which topical tretinoin repairs and prevents further photoaging is blocking induction of MMPs via AP-1. MAP (mitogen activated protein) kinase pathways transduce signals from growth factors, cytokines and other effectors of UVB damage. MAP kinase activation by low-dose UVB induces c-Jun, a component of transcription factor AP-1. Fisher et al.,25 showed that topical pre-treatment of human skin with tretinoin inhibits UV induction of c-Jun in human skin and consequently of AP-1 with blunting of MMP gene transcription. Ultimately, this prevents collagen degradation. The Michigan group also investigated the effect of UV irradiation on the regulation of pro-collagen synthesis in human skin in vivo, specifically the role of c-Jun, and asked if these effects could be reversed with tretinoin 0.1%.26 After a single UV exposure equivalent to 2 MEDs (minimal erythema doses), a significant reduction in type I and type III pro-collagen mRNA and protein was found. Multiple UV exposures of 1 to 4 MEDs prolonged the suppression of collagen synthesis. They then investigated the role of c-Jun in UV inhibition of type I pro-collagen gene expression in primary adult human skin fibroblasts. The fibroblasts were transfected with a CAT reporter gene under the control of type I (alpha-2) pro-collagen promoter, which contains the binding site for AP-1 (composed of Jun and Fos proteins), a negative regulator of pro-collagen transcription. UV irradiation rapidly induced c-Jun and reduced promoter activity. Lastly, the group pre-treated patients with 0.1% tretinoin and demonstrated preservation of type I and type III pro-collagen mRNA and protein levels, versus the reductions observed in UV-irradiated, vehicle treated skin. In summary, the above research has yielded several important lessons: • UV irradiation inhibits pro-collagen synthesis in human skin in vivo • Collagen synthesis is substantially reduced in chronically photoaged human skin in vivo • Tretinoin treatment of photoaged skin increases collagen synthesis • UV irradiation upregulates enzymes that degrade collagen in human skin in vivo, via induction of c-Jun, a component of the AP-1 transcription factor • Pre-treatment of skin with tretinoin inhibits UV induction of c-Jun and protects skin against loss of collagen. Telomeres and Aging Skin Why do we age? Let’s consider the possibility that aging represents an unintended and, until recently, largely irrelevant consequence of a highly effective cancer-prevention mechanism that has evolved over the millennia in multicellular higher organisms. This phenomenon centrally involves telomeres. It is a form of “antagonistic pleiotropy”27 that trades the protective effects of limited cellular proliferative capacity (no cancer) against its eventual detrimental effects, such as organ atrophy and poor wound healing. In a primitive world where humans rarely lived to age 40, both cancer and age-associated diseases were rare. As civilization outpaced evolution, however, “successful” members of society, those who lived for more than 40 years, began to experience problems due to failure of the mechanism (cancer) and simply to its existence (aging). Telomeres are tandem repeats of the DNA base sequence thymine-thymine-adenine-guanine-guanine-guanine (TTAGGG) and its paired complementary sequence at the end of each mammalian chromosome, consisting of 10,000 or so base pairs in humans. Because of the end replication problem, the inability of DNA polymerase to copy the final bases on each chromosome, with each round of cell division the telomere shortens. After a finite number of cell divisions (approximately 60 in human cells), the telomeres become “critically short” and the cell will no longer divide, regardless of the stimulation. In this way, telomeres function as the “biologic clock” and cause cells to enter a state termed replicative senescence.28 Structurally, the telomere is double stranded, except for a 3' guanine-rich single-stranded overhang that extends approximately 75 to 300 bases, repeats of TTAGGG. This overhang is normally concealed in a protective loop that is stabilized by binding proteins, the most important being telomeric repeat binding factor 2 (TRF2).29,30 If TRF2 is sequestered by a dominant negative construct, the telomere linearizes and the 3' overhang is digested, activating ATM (ataxia telangiectasia mutated protein, important for normal cell division and coordinating DNA-damage responses) and subsequently p53 (see diagram above). This leads to apoptosis in lymphocytes31 and senescence in human fibroblasts,30 known responses to acute DNA damage that also have been documented to occur with critical telomere shortening. Thus, experimental telomere loop disruption with TRF 2DN, replicative senescence after multiple rounds of cell division and acute DNA damage all cause identical responses: apoptosis in some cell types (such as lymphocytes) and permanent growth arrest in others (such as fibroblasts). Moreover, extensive studies have shown the apoptotic and senescent responses are mediated by identical proteins. Our group has shown that exogenous treatment of lymphocytes or fibroblasts with DNA oligonucleotides homologous to the telomere 3' overhang (T-oligos) mimics the effects of telomere disruption, causing these cells to undergo apoptosis or senescence, depending on cell type, mediated by ATM and the tumor suppressor protein p53.32 Treatment with T-oligos can trigger these and other DNA-damage like responses, including increased melanogenesis,33-35 cell cycle arrest35,36 and enhanced DNA repair capacity,37,38 in the absence of true DNA damage or telomere disruption. In combination, these data suggest that the 3' telomeric overhang sequence may be exposed by telomere shortening during chronologic aging or by acute DNA damage and that this event is mimicked by the exogenous treatment of cells with T-oligos. We hypothesize that exposure of the TTAGGG sequence activates a signaling pathway leading through ATM and p53 to transient growth arrest, apoptosis, adaptive differentiation or senescence (see diagram below). It further appears that the exact consequence of activating this pathway depends on cell type, intensity and duration of the initial signal. Skin experiences both intrinsic aging and chronic photodamage. The acknowledged overlap and mutual reinforcement of these processes, leading to “premature aging” or “photoaging,” might logically result from shared pathophysiologic mechanisms. The known and postulated roles of telomeres offer an attractive possible explanation for these observations (see diagram at left). Over many decades, epidermal stem cells, melanocytes and fibroblasts undergo multiple rounds of cell division with associated telomere shortening. In the event of UV irradiation or other environmental damage, additional compensatory cell division is likely. Simultaneously, cells are exposed to low-grade oxidative stress from normal cellular metabolism, stress again compounded by UV irradiation or other environmental exposures, leading to DNA damage, preferentially in the form of 8-oxo-guanine. UV irradiation further damages DNA through production of photoproducts, primarily cyclobutane pyrimidine dimers. Through telomere disruption and telomere-based DNA damage responses, these photo-products may upregulate DNA repair capacity and induce tanning or they may cause mutations in coding DNA that lead to cancer (see diagram below). Through telomere signaling, cells can also either senesce or undergo apoptosis, both protective anti-cancer responses. The proficiency and nature of these protective responses determines the character of photoaging. Adequate responses (possibly represented by skin types III/IV) allow people to tan well and to incur fewer mutations, whereas inadequate responses (possibly represented by skin types I/II) favor cell death or senescence, poor tanning, a high mutation rate and, ultimately, cancer. The ability of T-oligos to trigger such protective effects as enhanced melanogenesis (tanning), enhanced DNA repair capacity, reduced mutagenesis and enhanced antioxidant defenses, in the absence of DNA damage, suggests that unwanted aging/photoaging changes may someday be avoidable without disabling nature’s clever cancer prevention mechanism. Conclusion Much is now known about aging and photoaging at the physiologic and morphologic levels. Break-through studies over the past 15 years have partially elucidated the mechanisms by which one innate regulator of skin development and maintenance, tretinoin or at-RA influences these processes. The work has also provided our patients with a first generation of truly effective anti-aging therapies for their skin. Very recent research into the role of telomeres in regulating aging and responses to environmental DNA damage now provides a big picture in which to understand both intrinsic aging and photoaging. It is tantalizing to imagine that these new insights may also result in products to prevent and treat the unwanted consequences of skin aging.
A ging is a complex process dictated by an inherent genetic program and accelerated by environmental damage to genes and their protein products. Aging reduces maximal capacity and functional reserve of all organs in the body, ultimately to a point incompatible with life. This article reviews the recognized age-associated changes in skin and their pathophysiology and mechanism of treatment responsiveness, when known. It also explores current thoughts regarding the molecular basis of aging and its evolutionary value. Clinical Impact of Skin Aging Human skin is the body’s dynamic interface with the environment. As such, its roles include barrier function, chemical clearance, sensory perception, mechanical protection, wound healing, immune surveillance, thermoregulation, sebum production, vitamin D production and DNA repair.1 All of these functions decline with age, usually by 1/3 to 2/3 of young adult levels by the eighth decade. These losses predispose the elderly to a variety of minor and major health problems, both cutaneous and systemic, that include irritant contact dermatitis, intertrigo, skin cancer, chronic ulcers, heat stroke, hypothermia, osteomalacia and herpes zoster infection. In contrast to aging in most other body organs, however, skin aging impacts appearance and is of concern to individuals even when function is adequate to insure good health. The skin undergoes visible changes during life, which include those attributable to the passage of time alone (intrinsic aging) and superimposed changes due to the effects of chronic sun damage (extrinsic aging or photoaging) that are often far more pronounced and cosmetically disturbing. Intrinsic Aging of the Skin Clinically, aged skin is thin, pale, dry and wrinkled, with multiple benign neoplasms, such as seborrheic keratoses (see photo above).1,2 Hallmarks of aged skin, summarized in the table “Histologic Features of Aging Human Skin” on page 58, include epidermal and dermal atrophy with flattening of the dermal-epidermal junction (DEJ). This atrophy reflects a reduction in the proliferative keratinocyte population normally located in the rete ridges, in specialized epidermal cell populations, such as melanocytes and Langerhans cells, and loss of fibroblasts, blood vessels and appendages in the dermis. Ultimately, the changes at the DEJ impair cytokine-mediated communication as well as the transfer of nutrients and oxygen between dermis and epidermis.1 Overall, the dermis thins by 20% in the elderly,1 although thinning of intrinsically aged skin becomes most significant after the eighth decade.3 Collagen content per unit area of skin surface decreases by about 1% per year throughout adult life.4 Cultured skin fibroblasts have shown an age-associated increase in collagenase mRNA with a concomitant decrease in the rate of collagen synthesis.5 Dermal collagenase activity increases, leading to increased collagen degradation.6 Elastic fibers become fragmented with small cysts and lacunae, especially at the dermal-epidermal junction.7 There is a decrease in the overall vascularity of skin with age. Interestingly, in contrast to extrinsically aged skin, intrinsically aged skin has minimal pigmentary changes, although the density of melanocytes decreases progressively during adulthood by about 10% per decade.8 Extrinsic Aging of the Skin What accounts for the undesirable appearance of sun-damaged skin? A major contributing factor is the degradation of collagen and other components of the extracellular matrix (ECM). Type I collagen is a major component of dermal connective tissue, giving skin both strength and resiliency. Other components of the dermal matrix include type III collagen, elastin, proteoglycans and fibronectin. The best studied detrimental effects of sun-induced “premature skin aging” are destruction and disorganization of collagen. Increased release and activity of matrix metalloproteases (MMPs)9 and cytokines are induced by sun exposure, leading to increased collagen degradation. Cigarette smoking can compound the adverse effects of sun exposure, giving rise to coarse wrinkling and an increased risk of skin cancer.10-12 Additional features of sun-exposed skin include telangiectasias, prominence of pores, pre-cancerous lesions and a characteristic “bronzed” or chronically hyperpigmented appearance,1,2 associated with an increased density of melanocytes.8 The clinical and histologic characteristics of photoaged skin are summarized in “Features of Actinically Damaged Skin” on page 58. In contrast to sun-protected sites, the epidermis of sun-damaged skin may be either severely atrophic or hyperplastic.2 There are keratinocyte nuclear atypia and disturbed epidermal maturation,2 presenting clinically as actinic keratoses (AKs). Additionally, sun-damaged skin shows increased cellularity, prominent elastosis and decreased vascularity.13 A histologic hallmark of actinically damaged skin is elastosis,14,15 deposition of amorphous dermal elastin and collagen. In sun-protected skin, the amount of elastin increases slightly and also undergoes structural alterations,13,16,17 although these changes are subtle compared to those in actinically damaged skin. The clinical manifestations of photoaging depend on skin type and history of sun exposure. Skin types I and II tend to display freckling, likely reflecting mutational events, and eventually develop pre-cancerous lesions, such as AKs and non-melanoma and melanoma skin cancer. Phototype I or II skin also manifests proliferative exhaustion in the form of epidermal atrophy, focal depigmentation and pseudoscars (see photo on below). In constrast, skin types III and IV initially tan and later develop lentigines, epidermal thickening and other hypertrophic responses. In summary, patients with skin types I and II primarily undergo cell loss, mutation and dysplasia; while skin type III and IV patients tend to display protective hyperplasia. Retinoid Therapy Treatment of aging skin customarily involves the utilization of various cosmeceuticals, including hydroxy acids, growth factors, antioxidants, vitamins, hormones and retinoids.18 The scientific basis for the use of each class of skin products varies from very strong to non-existent. This section will review the best studied and only FDA-approved agent for the management of skin aging/photoaging: tretinoin (all trans-retinoic acid, at-RA), the active metabolite of vitamin A known to regulate embryogenesis and post-natal epithelial homeostasis at least in part by binding its nuclear receptors and modifying gene transcription.19 Tretinoin is a prescription drug sold under several brand names and generically. Retinol, which cells can metabolize to at-RA, is present in many cosmeceuticals, but controlled studies of its efficacy for aging/photoaging are not available. Early studies demonstrating the clinical and histologic efficacy of topical tretinoin for the treatment of photoaged skin were conducted in UV-irradiated hairless mice.20 In addition to the clinical amelioration of wrinkles in the mice, dermal changes included a “reconstruction zone”13 of new, structurally normal collagen in the papillary dermis.20 This zone of new collagen was later also demonstrated in human facial skin following topical tretinoin application.13 As described by the University of Michigan group who have performed many of the human studies, epidermal changes observed following application of tretinoin 0.05% to 0.1% to photoaged skin include increased cellular proliferation, a thickened granular layer, increased number of anchoring fibrils, and more normal appearing collagen in the papillary dermis.21 Rafal et al.,22 conducted a randomized, double-blinded study over a 10-month period on the efficacy of 0.1% tretinoin for the treatment of solar lentigines, another clinical manifestation of photodamage. The overall clinical response was evaluated by a single investigator in the majority of cases using degrees of color change. Clinical lightening was noted in 83% of treated facial lesions compared to 29% of controls. Pre-treatment and post-treatment histologic specimens were examined by a blinded observer using a semi-quantitative scale for various characteristics including epidermal pigmentation, spongiosis, stratum corneum compaction and dermal inflammation. Epidermal thickness was measured directly. The degree of epidermal pigmentation in the tretinoin group decreased by 35%, while it increased by 34% in the vehicle-treated group. To determine if topical application of tretinoin could reverse or repair effects of photodamage, particularly the degradation of collagen, Griffiths et al., used a mouse monoclonal IgG1 antibody that recognizes the aminopropeptide portion of pro-collagen I to provide an indirect measure of collagen I formation.23 Fifteen patients with photodamaged skin were treated for 10 to 12 months with tretinoin 0.1% daily, and biopsy specimens at baseline and at the conclusion of the study were compared and stained with the anti-body. Collagen I synthesis, which is reduced in photoaged skin, was partially restored with an increase of 80% over baseline values after tretinoin treatment, while vehicle control users experienced a 14% decrease. The finding of reduced levels of type I pro-collagen (indirect measure of collagen I) in photoaged skin was later verified along with reduced levels of type III pro-collagen.24 Matrix metalloproteases (MMPs) are proteolytic enzymes known to degrade collagen and components of the dermal ground substance. In a series of elegant experiments, Fisher et al., demonstrated that low-dose UVB irradiation of human skin in vivo induces matrix-degrading MMPs (collagenase, gelatinase, stromelysin) in a dose-dependent fashion.9 The genes encoding these inducible collagen and ECM degrading enzymes were up-regulated by the transcription factors AP-1 and NF-kB. In addition, AP-1 blocked collagen gene expression in dermal fibroblasts, impairing collagen synthesis. NF-kB amplified AP-1 and NF-kB release, resulting in further photodamage. Pre-treatment of human skin with topical tretinoin substantially blocked UVB induction of the transcription factor AP-1 binding to DNA and upregulation of mRNA, protein levels and activity for gelatinase and collagenase.9 The authors concluded that a major mechanism by which topical tretinoin repairs and prevents further photoaging is blocking induction of MMPs via AP-1. MAP (mitogen activated protein) kinase pathways transduce signals from growth factors, cytokines and other effectors of UVB damage. MAP kinase activation by low-dose UVB induces c-Jun, a component of transcription factor AP-1. Fisher et al.,25 showed that topical pre-treatment of human skin with tretinoin inhibits UV induction of c-Jun in human skin and consequently of AP-1 with blunting of MMP gene transcription. Ultimately, this prevents collagen degradation. The Michigan group also investigated the effect of UV irradiation on the regulation of pro-collagen synthesis in human skin in vivo, specifically the role of c-Jun, and asked if these effects could be reversed with tretinoin 0.1%.26 After a single UV exposure equivalent to 2 MEDs (minimal erythema doses), a significant reduction in type I and type III pro-collagen mRNA and protein was found. Multiple UV exposures of 1 to 4 MEDs prolonged the suppression of collagen synthesis. They then investigated the role of c-Jun in UV inhibition of type I pro-collagen gene expression in primary adult human skin fibroblasts. The fibroblasts were transfected with a CAT reporter gene under the control of type I (alpha-2) pro-collagen promoter, which contains the binding site for AP-1 (composed of Jun and Fos proteins), a negative regulator of pro-collagen transcription. UV irradiation rapidly induced c-Jun and reduced promoter activity. Lastly, the group pre-treated patients with 0.1% tretinoin and demonstrated preservation of type I and type III pro-collagen mRNA and protein levels, versus the reductions observed in UV-irradiated, vehicle treated skin. In summary, the above research has yielded several important lessons: • UV irradiation inhibits pro-collagen synthesis in human skin in vivo • Collagen synthesis is substantially reduced in chronically photoaged human skin in vivo • Tretinoin treatment of photoaged skin increases collagen synthesis • UV irradiation upregulates enzymes that degrade collagen in human skin in vivo, via induction of c-Jun, a component of the AP-1 transcription factor • Pre-treatment of skin with tretinoin inhibits UV induction of c-Jun and protects skin against loss of collagen. Telomeres and Aging Skin Why do we age? Let’s consider the possibility that aging represents an unintended and, until recently, largely irrelevant consequence of a highly effective cancer-prevention mechanism that has evolved over the millennia in multicellular higher organisms. This phenomenon centrally involves telomeres. It is a form of “antagonistic pleiotropy”27 that trades the protective effects of limited cellular proliferative capacity (no cancer) against its eventual detrimental effects, such as organ atrophy and poor wound healing. In a primitive world where humans rarely lived to age 40, both cancer and age-associated diseases were rare. As civilization outpaced evolution, however, “successful” members of society, those who lived for more than 40 years, began to experience problems due to failure of the mechanism (cancer) and simply to its existence (aging). Telomeres are tandem repeats of the DNA base sequence thymine-thymine-adenine-guanine-guanine-guanine (TTAGGG) and its paired complementary sequence at the end of each mammalian chromosome, consisting of 10,000 or so base pairs in humans. Because of the end replication problem, the inability of DNA polymerase to copy the final bases on each chromosome, with each round of cell division the telomere shortens. After a finite number of cell divisions (approximately 60 in human cells), the telomeres become “critically short” and the cell will no longer divide, regardless of the stimulation. In this way, telomeres function as the “biologic clock” and cause cells to enter a state termed replicative senescence.28 Structurally, the telomere is double stranded, except for a 3' guanine-rich single-stranded overhang that extends approximately 75 to 300 bases, repeats of TTAGGG. This overhang is normally concealed in a protective loop that is stabilized by binding proteins, the most important being telomeric repeat binding factor 2 (TRF2).29,30 If TRF2 is sequestered by a dominant negative construct, the telomere linearizes and the 3' overhang is digested, activating ATM (ataxia telangiectasia mutated protein, important for normal cell division and coordinating DNA-damage responses) and subsequently p53 (see diagram above). This leads to apoptosis in lymphocytes31 and senescence in human fibroblasts,30 known responses to acute DNA damage that also have been documented to occur with critical telomere shortening. Thus, experimental telomere loop disruption with TRF 2DN, replicative senescence after multiple rounds of cell division and acute DNA damage all cause identical responses: apoptosis in some cell types (such as lymphocytes) and permanent growth arrest in others (such as fibroblasts). Moreover, extensive studies have shown the apoptotic and senescent responses are mediated by identical proteins. Our group has shown that exogenous treatment of lymphocytes or fibroblasts with DNA oligonucleotides homologous to the telomere 3' overhang (T-oligos) mimics the effects of telomere disruption, causing these cells to undergo apoptosis or senescence, depending on cell type, mediated by ATM and the tumor suppressor protein p53.32 Treatment with T-oligos can trigger these and other DNA-damage like responses, including increased melanogenesis,33-35 cell cycle arrest35,36 and enhanced DNA repair capacity,37,38 in the absence of true DNA damage or telomere disruption. In combination, these data suggest that the 3' telomeric overhang sequence may be exposed by telomere shortening during chronologic aging or by acute DNA damage and that this event is mimicked by the exogenous treatment of cells with T-oligos. We hypothesize that exposure of the TTAGGG sequence activates a signaling pathway leading through ATM and p53 to transient growth arrest, apoptosis, adaptive differentiation or senescence (see diagram below). It further appears that the exact consequence of activating this pathway depends on cell type, intensity and duration of the initial signal. Skin experiences both intrinsic aging and chronic photodamage. The acknowledged overlap and mutual reinforcement of these processes, leading to “premature aging” or “photoaging,” might logically result from shared pathophysiologic mechanisms. The known and postulated roles of telomeres offer an attractive possible explanation for these observations (see diagram at left). Over many decades, epidermal stem cells, melanocytes and fibroblasts undergo multiple rounds of cell division with associated telomere shortening. In the event of UV irradiation or other environmental damage, additional compensatory cell division is likely. Simultaneously, cells are exposed to low-grade oxidative stress from normal cellular metabolism, stress again compounded by UV irradiation or other environmental exposures, leading to DNA damage, preferentially in the form of 8-oxo-guanine. UV irradiation further damages DNA through production of photoproducts, primarily cyclobutane pyrimidine dimers. Through telomere disruption and telomere-based DNA damage responses, these photo-products may upregulate DNA repair capacity and induce tanning or they may cause mutations in coding DNA that lead to cancer (see diagram below). Through telomere signaling, cells can also either senesce or undergo apoptosis, both protective anti-cancer responses. The proficiency and nature of these protective responses determines the character of photoaging. Adequate responses (possibly represented by skin types III/IV) allow people to tan well and to incur fewer mutations, whereas inadequate responses (possibly represented by skin types I/II) favor cell death or senescence, poor tanning, a high mutation rate and, ultimately, cancer. The ability of T-oligos to trigger such protective effects as enhanced melanogenesis (tanning), enhanced DNA repair capacity, reduced mutagenesis and enhanced antioxidant defenses, in the absence of DNA damage, suggests that unwanted aging/photoaging changes may someday be avoidable without disabling nature’s clever cancer prevention mechanism. Conclusion Much is now known about aging and photoaging at the physiologic and morphologic levels. Break-through studies over the past 15 years have partially elucidated the mechanisms by which one innate regulator of skin development and maintenance, tretinoin or at-RA influences these processes. The work has also provided our patients with a first generation of truly effective anti-aging therapies for their skin. Very recent research into the role of telomeres in regulating aging and responses to environmental DNA damage now provides a big picture in which to understand both intrinsic aging and photoaging. It is tantalizing to imagine that these new insights may also result in products to prevent and treat the unwanted consequences of skin aging.
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