Paolo U. Giacomoni
Aging is defined as the accumulation of molecular damages. Skin accumulates damages because of the mitochondrial production of superoxide in the course of phosphorylative oxidation, and because of the exposure to external damaging agents, such as solar radiation, gravitational traction, infections, electric fields, psychological stress, cigarette smoke, specific foods, anoxia and other agents.
All the factors contributing to the acceleration of the rate of accumulation of damages share the capability to induce the synthesis of I-CAM 1 in endothelial cells. This has prompted the proposal of the micro-inflammatory model of skin aging. According to this model, endothelial synthesis of I-CAM 1 allows the diapedesis of circulating monocytes, which enter the dermis and digest the surrounding extra-cellular matrix. During this process, nearby cells can be damaged and will secrete molecular signals to provoke vasodilation and diapedesis of more monocytes, to maintain the inflammatory cycle.
This model predicts the sagging and the thinning of the skin with age, the appearance of wrinkles, and the loss of elastic and rheological properties. This model does not account for the appearance of age spots.
With time, damages accumulate in organs and organisms, which lose some of the prerogatives characteristic of youth. Damages are chemical modifications to molecules in an organism. Molecular damages accumulating in cellular DNA are called somatic mutations. According to the gene where these mutations occur, the physiology of the cell or of the organ can change unremarkably or dramatically. It can even provoke the death of the cell, with minor consequences, or of the organism, which is a major consequence. Other biochemical damages can accumulate in cellular membranes or cell organelles, but the damages most relevant to the aging of an organism are the ones accumulating in the extra-cellular matrixes such as bones, blood vessels, dermis, or in postmitotic cells clustered in anatomical entities such as muscular fibers or fat tissues. Indeed, these damages are the ones more directly linked to the visible manifestations of the onset of the aging process.
The accumulation of damages consequent to the basal metabolism has been defined as natural or intrinsic aging (Giacomoni and Rein, 2004). The accumulation of damages provoked by the interaction with other environmental factors (exposure to humidity for the onset of osteoarthrosis) or by the way of life (lack of exercise for the aging of skeletal muscle) can be defined as accelerated or extrinsic aging. Expressions such as chronologic aging or premature aging are to be avoided. Aging is the continuous accumulation of damages with time and is therefore a chronologic phenomenon per se. It can't be premature because it starts at the fertilization of the egg. So it is recommended to use the expressions natural aging or accelerated aging. It is also true that genes can play a role in the overall rate of aging and in the life span of the individual. Indeed, the same environmental factor will have different effects on two individuals with different genetic backgrounds. To give an example, exposed to the same dose of UV radiation, individuals with a defective Glutathion-S-Transferease (GST) gene are more likely to develop skin cancers than individuals with a wild-type GST gene.
Aging has been defined as the accumulation of molecular damages over time (Novoseltsev et al., 2001; Giacomoni and D'Alessio, 1996b; Giacomoni, 1992). For a decade or so, biogerontologists have been discussing the necessity of aging. In particular the question has been asked about the selective advantage, if any, provided to a species by the fact that the individuals in the species undergo aging and therefore lose the capability to reproduce and to compete for food. Some scientists have elaborated a thought-provoking theory called disposable soma theory. According to this theory, the aging of the individuals represents a selective advantage for the species, and to ensure the aging of the individuals, specific genes called gerontogenes are turned on at a certain point in life. The function of gerontogenes is to trigger the process of aging, which eventually leads to the removal of the individual from competition.
In the last decade, though, gerontogenes were not found. Moreover it was observed that repair functions do not decrease with age (van Zeeland et al., 2001; Giacomoni et al., 2000) and no gene has been found that provokes the loss of functionality of cells or organs. Longevity in a species seems to be governed mainly by
Handbook of Models for Human Aging
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the capability to withstand aggressions and remove damages. The other possibility to achieve longevity is to live at a reduced metabolic pace by limiting the caloric intake, one of the consequences of which is to delay the onset of reproductive maturity.
For higher organisms, in particular humans, we can now say that the rate of aging is dictated mostly by way of life. The effects of caloric restriction on the process of aging are a good example in favor of the effect of lifestyle on the onset of aging (Giacomoni and Rein, 2001; Everitt, 2003). A Gedanken Experiment shows that, even in a hypothetical life without interaction with the environment other than water, sugar as food, and oxygen to breath, superoxide is generated in the course of oxidative phosphorylation, which provokes damages to cell constituents such as mitochondrial DNA (Giacomoni, 1992).
So, notwithstanding theoretical suggestions relative to genetically programmed aging (de Magalhaes 2003; Tan et al., 2001), consensus has been building in the scientific community about the understanding that the aging of the individual does not provide selective advantages to the species. The presently accepted conclusion is that the aging of a cell or of an organ, far from being an unavoidable curse, is more the consequence of its interaction with the environment (McArdle et al., 2002; Rattan, 2001).
Longevity and aging are two very different aspects of the life of an organism. Modifying longevity is not necessarily a relevant target in the scientific struggle against the undesirable effects of aging. If it were, dying young would be the best way not to grow old.
To study aging we need an organ accessible to analyses and such, that its aging does not bring prejudice to the survival of the organism (that is to say, one needs longevity to study aging).
Skin is one such organ. It is one of the largest organs in the human body. Its surface is smaller than that of intestinal epithelium, and its weight is smaller than that of the lungs, but with its two square meters and couple of kilograms, skin is respectable in size and mass. Human skin is vascularized, innervated, and immune-competent. It is able to secrete water and sebum. It supports the growth of muscle-associated hair, and human hair, at variance with other mammals, presents characteristics that differ according to the anatomical site where it grows. Indeed, the human hair growing on the scalp is morphologically and developmentally different from the human hair on the chest or on the pubis.
At variance with other mammals, human skin changes remarkably with aging. Hair on the scalp is lost or changes color, the epidermal surface undergoes discolorations, and the thickness of the dermis is reduced, particularly so in women. In the sites exposed to air and sun, the skin becomes elastotic (i.e., it loses elasticity because of the deposition of elastin), the capillary vessels are pushed farther away from under the basement membrane, sagging is generalized, and wrinkles appear on expression zones, which are those anatomical sites located on the human face that allow feelings to be expressed without words.
Histologically, the dermal-epidermal junction is flattened, possibly because of the constant stretching of the skin exerted by the gravitational field. The consequence of this flattening is an increased surface and hence the necessity to maintain a particular neurological state so that neural cells continuously send impulses to induce tendons and muscles to contract. In this way the formation of wrinkles allows the cutaneous tissue to remain confined to its natural location. Another consequence of this flattening is to reduce the apparent average epidermal thickness, and this has led some scholars to the incorrect belief that the number of epidermal layers is reduced with aging. The thickness of the stratum corneum and the duplication time of keratinocytes do not seem to undergo major changes with age. The immune response seems to be modified with aging, skin seems to increase its susceptibility to pruritus, and wounds require a longer time to heal.
The aging of the skin is more the consequence of way of life than of predetermination. Factors of skin aging such as solar radiation, cigarette smoke, tractional forces, electromagnetic fields, infections, psychological stressors, air pollutants, anoxia, wounds, and traumas have been identified. The genotype will play a role in the process of skin aging, too, because the same environmental insult, such as the same dose of ultraviolet radiation, will have different results on two individuals with differently active genes for DNA repair (Giacomoni et al., 2000; Heenen et al., 2001).
Visible signs on the surface of the skin are the consequence of time and environmental insults that trigger a series of cellular and physiological responses because of which, in the long term, the physical properties of the skin are modified. The role of solar radiation in skin aging can be understood when the necks of sailors are compared with the necks of nuns: Cutis rhomboidalis nuchae is characterized by profound textural and pigmentary changes, which occur upon chronic exposure to sun. Another consequence of excessive exposure to solar radiation is nodular elastosis or Favre-Racouchot disease, consisting of the appearance of cysts and comedons on the exposed parts of the skin. Other consequences of exposure to solar radiation are Millian's type citrine skin, teleangectasia, sagging, solar lentigo, and so on (for reviews on photo-aging, see Lyon and Fitzpatrick (1993) and Wlaschek et al. (2001)).
The skin of humans differs from the skin of other mammals, even rodents or mini-pigs. Yet for invasive laboratory studies, these two species have been selected, and now we know more about the effect of UV on the skin of hairless mice or of albino mini-pigs than on the skin of humans.
The physiological effects of ultraviolet radiation on skin has been analyzed by Bissett and coworkers (Bissett et al, 1989). Cohorts of laboratory rodents were exposed to quasi-monochromatic radiation at different wavelengths in the ultraviolet range. They observed that the sagging of skin is provoked by radiation at 365 nm. The ultra-structural details of the continuous exposure of mini-pig skin to artificial ultraviolet radiation have been described by Fourtanier and Berrebi (1989), who found that after 24 months of exposure to artificial UV radiation, the fibers of collagen are no longer oriented. They are entangled, instead, and this might explain the loss of elasticity, the loss of resilience, and the sagging of photo-aged skin. These findings, together with the observation that neutrophils infiltrates are to be found in the skin after moderate exposure to UV-B (Hawk et al., 1988) prompted the proposal that post UV-repair is analogous to wound healing (Giacomoni and D'Alessio, 1996a). Indeed wound-healing and post-UV-repair seem to share common features, in particular the mechanisms of tissue modifications, which include massive cell migration, proliferation, phenotypic differentiation, and enhanced biosynthetic activities. Macrophages and fibroblasts express embryonic fibronectin during cutaneous wound-healing, thus providing an extra-cellular matrix that facilitates wound repair. They are able to promote cell migration so that subpopulations of fibroblasts able to secrete migration-stimulating factors (MSF) might undergo transient and local expansion during wound healing. The process of disassembly of the extra-cellular matrix (ECM) operates via collagenases, proteoglycanases, and other lythic enzymes of macrophage origin. The last phase of wound healing involves a systematic dissolution of the granulation tissue, which is accompanied by a gradual loss of cells and vasculature and by exhaustive restructuring of the ECM. Newly synthesized ECM molecules do not fit, per se, into an ordered structure but can modulate the original geometric framework, which is essential for cell-matrix interaction. In fact, the disordered structure, which is observed upon chronic UV irradiation, might well be the consequence of one particular disposition of newly synthesized molecules. This is to say that to improve skin repair and fight skin aging, we should not try and accelerate the synthesis of collagen, because the turnover of collagen in human skin is of the order of 15 years or more (Verzeijl et al., 2000). To improve skin repair we might well have larger chances of success by trying and looking for treatments able to help in maintaining newly synthesized macromolecules in an orderly structure. This, at least in laboratory rodents, seems to be, possible by specifically inhibiting the maturation of TGF-^ (Shah et al., 1992). It might be interesting to notice that TGF-^ is an agent often used in cosmetic products to stimulate the synthesis of collagen. The results by Shah et al. (1992) might be overly interesting if the "quantity" of collagen production were in conflict with the "order" of the structure of the macromolecular fibers.
The research leading to the conception of the micro-inflammatory model of skin aging (Giacomoni and D'Alessio, 1996b) was prompted by the understanding of the relevant role played by actors of the inflammatory response in wound-healing and in post-UV-repair. In the course of such research, it was noticed that all the commonly accepted accelerators of the aging process shared a common feature the capability to induce the synthesis of ICAM-1 in the endothelium. This meant that all the factors recognized as being able to accelerate skin aging had the capability to trigger a self-maintained inflammatory response.
Indeed, upon synthesis of ICAM-1 in the endothelium, circulating monocytes secrete hydrogen peroxide to perform diapedesis across the endothelium lining the vascular wall. Once in the dermis, the macrophages secrete lythic enzymes and reactive oxygen species (ROS) to fray a path in the dermis.
In the majority of instances, macrophages respond to chemotactic signals to reach and destroy infectious agents, or to remove damaged skin cells. In some instances, as in the case of anoxia-generated diapedesis, macrophages in the dermis release collagenases and ROS so as to damage the smooth muscle surrounding veins. When anoxia is repetitive, as in the case of the veins in the legs of workers obliged to long-lasting stasis, this provokes such damage to smooth muscles that the vein becomes varicous.
In these conditions, it is highly likely that nearby resident cells, such as fibroblasts or keratinocytes, are damaged by the free radicals. When this happens, the damage can trigger the cascade of arachidonic acid and secretion of prostaglandines and leukotrienes, which diffuse to interact with resident mast cells. Upon binding of these mediators to specific receptors, the resident mast cells release histamine and TNF-a, which in turn stimulate the release of P-selectins and the neo-synthesis of ICAM-1 in endothelial cells. The cycle therefore is closed, and the micro-inflammatory status is maintained.
Does this model account for all the factors of skin aging and for all the phenomenological characteristics of aged skin? Some of the accepted factors of skin aging are direct cell- or tissue-damaging factors. When damage to cells or tissue is generated (e.g., by UV-radiation, smoke-related free radicals, infectious agents, or wounds and traumas), thousands of damages are provoked to the ECM, resident cells, and vessel walls by the free radicals and lythic enzymes, which are released in the course of the inflammatory response, consequent to the diffusion of cytokines produced via the arachidonic acid cascade. What about the other factors of aging that are not directly damaging agents? Traction and gravitational forces provoke the activation of phospholipase A2, an enzyme involved in the arachidonic acid cascade. Anoxia induces ICAM-1 synthesis and diapedesis of macrophages, which start digesting the ECM around veins or other blood vessels. Glucose binds to proteins in a non-enzymatic glycation process, and glycated proteins are inducers of ICAM-1 synthesis. Electromagnetic fields associated with computers provoke the release of histamine, IL-1 and IL-6. (A summary of the results of scientists at the Karolinska Institute of Stockholm on this subject is given by Giacomoni and Rein, 2001.) Neuro-peptides regulate the expression of cell-adhesion molecules on both leukocytes and endothelial cells in a coordinated effort to control neurogenic inflammation. These phenomena trigger a cycle of self-maintained inflammatory response, which comprises the induction of the neo-synthesis of ICAM-1, and are summarized in Giacomoni and Rein (2001).
This micro-inflammatory model of skin aging emphasizes the aging of the connective tissue and of the extra-cellular matrix. Macroscopic consequences of this model are verified by the experience. The recognition that post-UV-repair and wound-healing share the ECM remodeling as a common feature has allowed us to understand why blood vessels are deeper down in aged skin than they are in young skin. The sagging of the dermis is the consequence of a modified ECM and is accompanied by an overall increase of the surface of the skin, particularly of the face.
It can be noted at this point that muscular and neurological actions are involved in the formation of wrinkles to maintain the skin with increased surface to cover a skull. This was first observed by neurophysiol-ogists who noticed that in the case of hemiplegia, the wrinkles were observed only on that part of the forehead that was not paralyzed (Prunieras, private communication). Surgeons also noticed that patients subjected to anesthesia did not display wrinkles (Marty, private communication).
The increase of skin surface and the reduction of body volume can be invoked to explain the observation that the thickness of the epidermis is diminished with aging. This is more the consequence of the stretching of the skin than of a modification of the turnover rate of the keratinocytes. Indeed, the turnover of the keratino-cytes does not change much with aging; this is witnessed by the fact that the thickness of the stratum corneum does not change with aging (Gilchrest, 1993).
On the other hand, the micro-inflammatory model of skin aging does not provide detailed explanations for the appearance of other visible signs of aging associated with the surface of the skin, such as discoloration and dryness.
It is known that a permanent state of irritation (i.e., with constantly high levels of mediators of irritation such as IL-1) stimulates the production of melanin by the melanocytes. In this case the micro-inflammatory model of skin aging predicts that with time, the skin should undergo a progressive homogeneous darkening. What the model fails to predict is the appearance on specific skin regions, of the so-called age spots, dark regions of less than 0.25 cm2 in sun-exposed areas, the color of which has been suggested to be the consequence of the accumulation of melanin and/or lipofuschin. Recent studies have demonstrated a role for the growth factor, insulin-like growth factor I (IGF), in the appearance of the signs of aging.
It can be noted at this point that elderly men with low serum IGF levels (Bonafe et al., 2003) are more likely to be healthy (van den Beld et al., 2003). Furthermore, long-lived individuals have lower levels of the IGF receptor (Bonafe et al., 2003). Higher levels of IGF appear to be pro-inflammatory. It has been long known that IGF induces histamine release from baso-phils in inflammatory bronchial asthma (Morita et al., 1993). More recent evidence indicates IGF plays an important regulatory role in inflammatory processes. Shortly after injury, acutely inflamed human wounds show increases in IGF-1 protein concentrations, believed to arise from blood sources (Wagner et al., 2003). Similarly, IGF is up-regulated following tendon injury, where its expression in increased during the early inflammatory phase (Molloy et al., 2003). In this animal model, IGF is believed to mediate the proliferation and migration of fibroblasts. The actions of IGF and other growth factors in promoting aging and inflammatory processes are further regulated by neuro-peptides. Neuropeptides are now being considered in the aging process. A recent study in normal children demonstrated age-dependent changes in serum levels of several neuropeptides, as well as IGF (O'Dorisio et al., 2002). Of particular interest here is that that the physiological actions of neuropeptides are synergistic with IGF. Although this has mostly been demonstrated using cultured neurons (Jones et al., 2003), recent studies with epithelial wound healing demonstrate a similar effect with substance P and IGF (Nagano et al., 2003; Nakamura et al., 2003). These results demonstrate the complexity of the regulatory mechanisms involving neuropeptides in the process of skin aging. Although not yet proven, these mechanisms are also thought to affect the activation of melanocytes, which is possibly linked to the age-associated skin discoloration.
Skin dryness is a more complex state of affairs, because the model does not provide suggestions to predict the accumulation of water or the synthesis of lipids in the stratum corneum. This parameter depends so closely from yearly cycles (seasons) and meteorological conditions (temperature, relative humidity) that the individual and local variability provide a "noise" able to mask the trends dictated by aging (if any). Hormonal imbalance and the loss of capability to retain water in the dermis, which occur at the onset of menopause, are not predicted by the micro-inflammatory model of skin aging, and correctly so because the onset of menopause is more a development- than an aging-related phenomenon. This being said, we could add that the moisturization of the skin is difficult to assess both instrumentally and via self-description. When the assessment of skin moistur-ization is conducted by self-description, the words used by different subjects do not mean the same thing to everyone. It is now felt, for instance, that the word "moisturization" is used to describe a state of suppleness, flexibility, and softness, more than an actual state of hydration. Instruments to assess the hydration of the skin measure either the gradient of the concentration of water vapor in the proximity of the skin surface, or the conductivity of the surface of the skin. In both cases the correspondence between the state of the skin and the experimental results is not unequivocal. Indeed, a high value of the gradient of concentration of water vapor (called trans-epidermal water loss, or TEWL) can be the consequence of a wellhydrated skin or of a lack of surface lipids. Similarly, high conductivity can be the consequence of high water content or of low water content concomitant with high electrolyte content in the surface of the skin. So when we measure macroscopic properties of the skin such as TEWL versus age, we observe that at young age the TEWL is low, then it increases during young adulthood and maturity, and it decreases again in old age (Giacomoni et al., 2002). From these measurements it is impossible to conclude that young skin is hydrated and has a good barrier, whereas old skin is dry and has a poor barrier. In the absence of data that can be unequivocally interpreted, the comparison of the predictions with the experimental results is a useless endeavor.
The micro-inflammatory model of skin aging provides a sound mechanistic paradigm to understand the aging of the dermis and of the extra-cellular matrix in general. The model also recognizes new factors of aging, insofar as it may "predict" whether a particular aggression will be able to trigger the synthesis of ICAM-1 in the endothelium. It also allows us to consider interventions to reduce the rate of aging, and in this respect it can be considered a successful model. It has also been applied to describe the aging of other organs of the human body, such as joints (Pincus, 2001) and the brain
(Wilson et al., 2002), and for the understanding of the onset of age-associated diseases such as osteoarthritis and senile dementia. The micro-inflammatory model of skin aging, on the other hand, fails to predict the appearance of age-related modifications of the surface of the skin.
As of today, mainly because of the lack of appropriate experimental devices able to provide unequivocal data, the micro-inflammatory model of skin aging cannot be applied to predicting changes in the production of those molecules that are essential for maintaining the hydro-dynamic equilibrium of the surface of the skin and that seem to be impaired with aging. The micro-inflammatory model fails to predict the appearance of age spots. This might be the consequence of our ignorance relative to the ontogenesis of these spots. We do surmise a role for resident neural-crest melanocytes, which respond to neuropeptides (Kauser et al., 2003); this has been suggested in the overall process of skin aging (Giacomoni and Rein, 2001).
The micro-inflammatory model of skin aging has prompted the use of anti-oxidant and anti-inflammatory agents in creams and ointments aimed at reducing the rate of aging of the connective tissue via the topical applications of such ingredients. The rationale behind these experiments was that anti-oxidants and anti-inflammatory agents are expected to reduce the rate of accumulation of damages provoked in the course of a micro-inflammation subsequent to the synthesis of ICAM-1. As of today, positive preliminary results are now and then communicated to dermatology or cosmetic congresses, but published evidence is yet to be found.
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