Light and scanning electron microscopic studies on wrinkles in aged persons' skin

Wrinkles in six aged persons (67–82 years of age) have been investigated by light microscopy (LM) and scanning electron microscopy (SEM). There are two types of wrinkles. One is a deep wrinkle which develops on the sun-exposed skin and does not disappear on stretching (permanent wrinkle). The LM and SEM showed less elastotic change in the upper dermis in the area of wrinkle than in that of the surroundings. The other type is a shallow wrinkle which develops on sun-protected skin and disappears on stretching (temporary wrinkle). The LM and SEM showed the decrease or loss of the elastic fibres in the papillary dermis as seen in ageing skin.     

The Elastic Tissue of the Skin

In order to separate the changes of actinic damage from those of simple aging, we studied the elastic fibers in low and high sun-exposed skins of normal subjects at different ages. Low sun-exposed skin shows chronologic aging lesions only. These begin at age 30 with a disappearance of oxytalan fibers and with some abnormalities in the reticular and deep dermis; at age 40, aging changes are established: no oxytalan fibers, marked abnormalities, and lysis of elaunic and elastic fibers. In high sun-exposed skin, age-related lesions also occur but are associated with more or less precocious elastotic degeneration in reticular and deep dermis. Both types of aging fibers are revealed by the antielastin antibody HB 8, disappear with elastase, but resist collagenase. Actinic elastosis clearly originates from elastin. The two types of change differ in electron microscopic appearance: with spontaneous aging, elastic fibers are disintegrated (loose and porous fibers); in actinic damage, elastotic fibers are thicker and have accentuated microfibril dense masses. The age-associated lesions could be due to the activity of protease of fibroblastic origin whereas the elastotic degeneration is probably due to the actinic stimulation of fibroblasts.     

From skin microrelief to wrinkles. An area ripe for investigation

Skin microrelief alters progressively with age. Wrinkles do not result from these changes but are superimposed upon them. Wrinkles result from structural changes in the epidermis, dermis and hypodermis. Four types of wrinkles can be recognized. Type 1 wrinkles are atrophic. Type 2 wrinkles are elastotic. Type 3 wrinkles are expressional. Type 4 wrinkles are gravitational. Each type of wrinkle is characterized by distinct microanatomical changes and each type of wrinkle develops in specific skin regions. Each is likely to respond differently to treatment. Skin microrelief and skin folds can be identified on histological examination. By contrast, only minimal dermal changes are found beneath permanent or reducible wrinkles compared with immediately adjacent skin. A series of objective and non-invasive methods is available to quantify the severity of wrinkling.     

Morphological study of the relationship between solar elastosis and the development of wrinkles on the forehead and lateral canthus

OBJECTIVE To identify whether there is a relationship between solar elastosis and the development of wrinkles in human skin. DESIGN Wrinkle depth was measured on the forehead and lateral canthus of human cadavers using image analysis. The thickness of the dermis was measured in skin sections obtained around wrinkles and stained with Elastica-van Gieson. SETTING Gross Anatomy Section, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. SUBJECTS Fifty-eight male and female cadavers (age range at death, 29-93 years). MAIN OUTCOME MEASURES The ratio of solar elastosis dermal thickness to full dermal thickness (elastosis ratio) was calculated and compared between the deepest point of a wrinkle (wrinkle point) and a point within 1 mm where no wrinkle existed (nonwrinkle point). The relationship between elastosis ratios and wrinkle depths was investigated. RESULTS Advanced solar elastosis was present at nonwrinkle points but was present a little bit at wrinkle points. On the forehead, a positive correlation between elastosis ratios and wrinkle depths was observed at nonwrinkle points but not at wrinkle points. On the lateral canthus, a positive correlation between elastosis ratios and wrinkle depths was observed at nonwrinkle points, as well as at wrinkle points until the wrinkle became deeper than one-half of the original dermal thickness (0.6 mm). Solar elastosis on the lateral canthus ceased developing at this point, but the wrinkle developed further. CONCLUSIONS Solar elastosis tends to commence with the development of a wrinkle until the wrinkle becomes deeper than 0.6 mm. This tendency is less evident at wrinkle points than at nonwrinkle points.     

Immunohistochemical study of chronological and photo-induced aging skins using the antibody raised against D-aspartyl residue-containing peptide

BACKGROUND: Biologically uncommon D-aspartyl residues have been reported in the elderly tissues such as tooth, eye lens, aorta, and brain. We have previously prepared the antibody against D-aspartyl residue-containing peptide and found that it reacted with elastotic material of actinic elastosis. METHODS: Immunoreactivity of the normal skins obtained from sun-exposed and sun-protected skins of varied ages with this antibody was studied. RESULTS: In the sun-exposed skins, the antibody showed negative reaction with the skin specimens of young donors, whereas it reacted with elastotic materials of actinic elastosis of the elderly. In the sun-protected skins, the antibody recognized elastic fiber-like structures and inner layer of vessels found from the mid to lower dermis of old donors but showed no positive reaction to skin specimens of young donors. CONCLUSIONS: The results suggest that the antibody is a potent marker for chronological and ultraviolet (UV)-induced skin aging. Unusual eosinophilic bodies seen in the superficial dermis in the sun-exposed area of the elderly skins were also immunoreactive with the antibody, suggesting that the eosinophilic bodies resulted from UV-induced skin damage.     

N(ɛ)-(carboxymethyl)lysine modification of elastin alters its biological properties: implications for the accumulation of abnormal elastic fibers in actinic elastosis

Accumulation of degenerated elastic fibers in the sun-exposed skin designated as actinic elastosis is a histological hallmark of photodamaged skin. Previous studies have indicated that the elastic fibers of actinic elastosis interact with lysozyme and are modified by N(?)-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGEs). We studied here how CML modification of elastin is involved in the pathogenesis of actinic elastosis. The CML-modified insoluble elastin became resistant to neutrophil elastase digestion, which was reversed by treatment with aminoguanidine, a potent inhibitor of AGE formation. In a temperature-dependent aggregation assay, CML-modified elastin rapidly formed self-aggregates, the size of which was larger than unmodified elastin. The elastic fiber sheets prepared from CML-modified α-elastin showed 3D wider diameter, tortuous appearance, and decreased elasticity on tensile tests. The CML-modified α-elastin, but not unmodified α-elastin, was found to bind to lysozyme in vitro, supporting the immunohistochemical findings that the antibodies for lysozyme and CML reacted simultaneously with the elastic fibers of actinic elastosis and UV-irradiated skin. The glycated elastin is likely to cause the accumulation of abnormally aggregated elastic fibers and unusual interaction with lysozyme in actinic elastosis.     

Immunohistochemical detection of lipid peroxidation products, protein-bound acrolein and 4-hydroxynonenal protein adducts, in actinic elastosis of photodamaged skin

Abstract Acrolein and 4-hydroxy-2-nonenal (HNE) are both byproducts of a lipid peroxidation reaction. Actinic elastosis in photodamaged skin of aged individuals is characterized by the accumulation of fragmented elastic fibers in the sun-exposed areas. To study whether a lipid peroxidation reaction is involved in the accumulation of altered elastic fibers in actinic elastosis, skin specimens obtained from sun-damaged areas were immunohistochemically examined using the antibodies against acrolein and HNE. Both antibodies were found to react with the accumulations of elastic material. Double immunofluorescence labeling demonstrated that acrolein/elastin and HNE/elastin were colocalized in the actinic elastosis. Western blot analysis showed that the polypeptide with a molecular weight of 62 kDa reacted with anti-acrolein, anti-HNE and anti-elastin antibodies. The results suggest that acrolein and HNE may be associated with actinic elastosis. Received: 18 October 2000 / Revised: 3 February 2001 / Accepted: 27 April 2001     

Pinguecula and Actinic Elastosis

Three cases of pinguecula (conjunctival elastosis) were ultrastructurally investigated. Findings were compared with the features described in cutaneous actinic elastosis. Changes observed were not uniform. In both diseases, numerous elastotoc fibers were present with a finely granular matrix and masses of dense grains. The degenerative changes of the elastotic fibers evolved differently in conjunctival stroma and in dermis. The superficial extracellular concretions observed in pinguecula seemed to be an ultimate stage of elastotic degeneration. Like actinic elastosis and elastosis observed in chronic radiodermatitis, pinguecula is believed to result from a dystrophic increased elastogenesis induced by chronic irradiation, with secondary degenerative changes.     

Expression of elastin-related proteins and matrix metalloproteinases in actinic elastosis of sun-damaged skin

Abstract Actinic elastosis is characterized by an accumulation of elastotic material in the upper dermis and is considered to be a manifestation of ultraviolet-induced skin aging. To compare the structural components of the elastotic material in actinic elastosis with those in normal skin, skin specimens were stained with antibodies raised against various elastin-related proteins. Elastotic materials exhibited a strong reaction to the antibodies for elastin, microfibril-associated glycoprotein-1 (MAGP-1), MAGP-4, matrix metalloproteinase 1 (MMP-1), MMP-2 and MMP-3, but a diminished reaction to anti-MMP-9 antibody. Fibroblast cell lines from the upper dermis of affected and unaffected skin were established, and the mRNA levels of MMPs were determined. MMP-1 and -2 mRNA levels were found to be elevated approximately twofold in the fibroblasts from actinic elastosis. Since MMP-1 and -2 are considered to be major enzymes involved in the degradation of matrix components, the accumulation of elastotic materials in actinic elastosis may be related to the degradation process. Received: 3 March 1999 / Received after revision: 26 August 1999 / Accepted: 6 September 1999     

Unusual wrinkle formation after temporary skin fixation followed by UVB irradiation in hairless mouse skin

Abstract Generally, many wrinkles form on the human face, and temporary wrinkles eventually become permanent. We evaluated the effects of temporary skin fixation on wrinkle formation after UVB irradiation using the back skin of hairless mice. In the group treated with UVB irradiation immediately after production using cyanoacrylate resin of an artificial groove parallel to the midline, wrinkles formed parallel to the midline, an uncommon direction for wrinkle formation in this mouse model. These wrinkles did not disappear even when the skin was stretched. No such changes were observed in the group in which only the temporary groove alone was produced without UVB irradiation. In 3-D surface parameter analysis, all roughness parameters in the group treated with UVB irradiation immediately after production of an artificial groove were significantly increased relative to the age-matched control group. In contrast, no differences were observed between the group in which only the temporary groove alone was produced without UVB irradiation and age-matched controls. The results of this study suggest that both a temporary groove in the skin and UVB irradiation are necessary for wrinkle formation in the back skin of hairless mice.     

Presence of Merkel cells in sun-exposed and not sun-exposed skin: a quantitative study

Summary Merkel cells (MCs), the neuroendocrine cells of the skin cannot be identified with certainty using conventional light microscopic staining methods. Using immunoperoxidase microscopy with antibodies specific for cytokeratin 18, which has been established as a marker protein of MCs, we have evaluated the numbers of MCs per mm² skin in normal and sun-damaged upper arm skin. The sun-exposed skin contained twice as many MCs as the not sun exposed skin. Further quantification of MC density at various body sites (trunk, leg) showed a rather variable but often unexpectedly high MC density. The possible role of MC in development of actinic elastosis is discussed.     

光线性弹性组织变性及其机制

光线性弹性组织变性为皮肤光老化的一部分,在组织学上主要表现为弹性纤维结构和功能的异常.目前,其机制尚不完全清楚,其中,紫外线起着重要的作用.其可通过影响基质金属蛋白酶、弹性蛋白酶、簇连蛋白等多种酶和蛋白的表达,导致弹性蛋白和微原纤维等弹性纤维成分发生改变,并促使光线性弹性组织变性的形成和发展.此外,非酶糖化反应、红外线及热休克反应在光线性弹性组织变性中也发挥一定的作用.     

Actinic elastosis in black skin

Summary In 9 otherwise dermatologically normal South African Black volunteers (1–72 years old), sunlight-exposed and non-exposed skin has been examined by light- and electronmicroscopy with special references to age-dependent and actinic alteration of dermal structures. Two 72 year old Blacks exhibited typical dermal elastosis: in 1 case to a marked extent and already detectable by lightmicroscopy, in the other case only to a mild degree. In contrast, only age-related elastic fibers were revealed in the lower dermis of sunlight-exposed skin and in all dermal layers of unexposed skin. These observations are contradictory to the general view that actinic elastosis does not occur in Blacks. The elastotic material is obviously a de-novo synthesized and secreted pathological product of chronically UV-altered fibroblasts.Reported in part at the XV. International Congress of Dermatology, Mexico City, October 1977, by M. Dogliotti, W. Ch. Marsch, E. Schober and F. Nürnberger     

Increased deposition of fibulin-2 in solar elastosis and its colocalization with elastic fibres

BACKGROUND: Fibulin-2 is a 195-kDa protein belonging to a novel family of extracellular matrix proteins that might be involved in microfibril and elastic fibre organization. OBJECTIVES: To determine the localization of fibulin-2 in relation to elastic fibres in normal skin and in solar elastosis characterized by increased elastotic material in the papillary dermis. METHODS: The expression and synthesis of fibulin-2 was investigated by means of in situ hybridization, immunohistochemistry and Western blot analysis in normal and photoaged skin. RESULTS: Immunohistochemistry and elastic tissue staining revealed that fibulin-2 deposition mainly colocalized with microfibrils and elastin fibres, with a marked staining of elastotic material in solar elastosis. Western blot analysis demonstrated that in photoaged skin fibulin-2 showed the same electrophoretic mobility as in sun-protected skin. However, in actinic elastosis the amount of fibulin-2 was significantly higher. In addition, smaller degradation products were detectable, presumably reflecting increased proteinase activity in photodamaged skin. CONCLUSIONS: This study shows that deposition of fibulin-2 and elastin is highly co-ordinated, indicating that this protein plays an important role in elastic fibre and microfibril formation in normal and actinically damaged skin.     

Glycation during human dermal intrinsic and actinic ageing: an in vivo and in vitro model study

BACKGROUND: Non-enzymatic glycation occurring in normal human skin plays an important part in ageing. OBJECTIVES To visualize and quantify, in human subjects, the extent of glycation during human dermal intrinsic and actinic ageing, and to develop a reliable reproducible in vitro model for evaluating the efficacy of potential inhibitors of glycation. METHODS: By immunohistochemistry using a monoclonal antibody recognizing carboxymethyl lysine, an advanced glycation end-product (AGE) (first objective), and by incubating dead de-epidermized dermis (DED) with glucose to simulate ageing-induced glycation in a human dermal equivalent model (second objective). RESULTS: We found that glycation of the dermis generally arises after 35 years, then increases rapidly with intrinsic ageing. We also noticed an enhancement of glycation by solar irradiation that occurred via glycation of the elastic fibre network or solar elastosis tissue. In the model, production of AGEs appeared in a time-dependent way, mimicking glycation observed in vivo during chronological ageing. Irradiation of DED before incubation with glucose strongly enhanced induction of AGEs, corresponding to the effect of solar irradiation on AGEs observed in vivo. CONCLUSIONS: These results confirm a marked increase of AGEs during intrinsic ageing in normal human skin and also suggest that glycation is enhanced in photoaged skin.     

Selective inhibition of skin fibroblast elastase elicits a concentration-dependent prevention of ultraviolet B-induced wrinkle formation

We previously reported that wrinkle formation in the skin following long-term ultraviolet B irradiation is accompanied by decreases in skin elasticity and the curling of elastic fibers in the dermis. We further showed that wrinkles could be repaired by treatment with retinoic acid and that this was concomitant with the recovery of skin elasticity ascribed to the repair of damaged elastic fibers. Those studies suggested that decreasing the tortuosity of dermal elastic fibers is an important factor involved in inhibiting or repairing wrinkle formation. Therefore, it is of particular interest to determine whether the inhibition of elastase activity in vivo would prevent the damage of dermal elastic fibers and might abolish wrinkle formation associated with the loss of skin elasticity. Because the major elastase in the skin under noninflammatory conditions is skin fibroblast elastase, we used a specific inhibitor of that enzyme to assess its biologic role in wrinkle formation. The hind limb skins of Sprague-Dawley rats were irradiated with ultraviolet B at a suberythemal dose three times a week for 6 wk. During that period, 0.1-10.0 mM N-phenetylphosphonyl-leucyl-tryptophane, an inhibitor of skin fibroblast elastase, was applied topically five times a week. N-phenetylphosphonyl-leucyl-tryptophane application at concentrations of 0.1-1.0 mM abolished wrinkle formation in a concentration-dependent manner, with a peak for inhibition at 1.0 mM. This inhibition was accompanied by a continued low tortuosity of dermal elastic fibers and a maintenance of skin elasticity. Measurement of elastase activity after 6 wk of ultraviolet B irradiation demonstrated that whereas phosphoramidon-sensitive elastase activity was significantly enhanced in the ultraviolet B-exposed skin, there was no significant increase in that activity in the ultraviolet B-exposed, N-phenetylphosphonyl-leucyl-tryptophane-treated skin. These findings suggest that skin fibroblast elastase plays an essential part in the degeneration and/or tortuosity of elastic fibers induced by cumulative ultraviolet B irradiation.     

Fibulin-5 deposition in human skin: decrease with ageing and ultraviolet B exposure and increase in solar elastosis

BACKGROUND: Fibulin-5 was recently found as a secreted extracellular matrix protein that functions as a scaffold for elastic fibres. However, the distribution of fibulin-5 in human skin and its changes during the ageing process are not known. OBJECTIVES: To explore the involvement of fibulin-5 in skin ageing, the age-dependent changes in fibulin-5 localization in human skin were examined compared with those of other elastic fibre components including elastin, fibrillin-1 and fibulin-2. Methods The distribution of elastin, fibrillin-1, fibrillin-2, fibulin-2 and fibulin-5 was investigated by means of immunohistochemistry using their specific antibodies. Skin samples were recovered from 12 healthy subjects undergoing plastic surgery. Ultraviolet (UV) B-irradiated or control nonirradiated buttock skin samples were obtained from two healthy volunteers at 2 days after the irradiation at 2 minimal erythemal doses. RESULTS: In the reticular dermis of young sun-protected skin from the upper arm, fibulin-5 colocalized with the other elastic fibre components, while in the papillary dermis fibulin-5 showed candelabra-like structures perpendicular to the epidermis with an unstained area just beneath the epidermis, which was similar to that of elastin but not fibrillin-1. Fibulin-5 in the reticular dermis decreased and disappeared with age even in sun-protected skin from the thigh, abdomen and upper arm. In sun-exposed skin, fibulin-5 was extremely reduced in the dermis of cheek skin even from a 20-year-old man. UVB irradiation reduced fibulin-5, fibulin-2 and elastin markedly, moderately and weakly, respectively, compared with levels in control nontreated skin. Interestingly, the deposition of fibulin-5 was increased in solar elastosis, like that of other elastic fibre components. CONCLUSIONS: These results suggest that fibulin-5 is a good marker of skin ageing and that the earlier loss of fibulin-5 may involve age-dependent changes in other elastic fibre components.     

Elastosis in chronic radiodermatitis AN ULTRASTRUCTURAL STUDY

The electron microscopic study of six cases of chronic radiodermatitis from sun-exposed and unexposed skin, confirms the observation of elastosis previously described with light microscopy. The pathological findings are comparable with those found in actinic elastosis; increased width of the fibres, irregularity of their shape, increased density and granularity of the microfibrils and holes with many lobes in the matrix. The findings may be due to transformation of elastic fibres into elastotic fibres or the formation of dystrophic elastotic fibres by dermal fibroblasts.     

Elastic tissue changes in generalized granuloma annulare

The histological picture in granuloma annulare (GA) is well defined. The types of infiltrate and the changes in collagen and elastic tissue have been repeatedly described. This is a preliminary study focused on the elastic tissue changes in lesions of generalized GA. Biopsies from four patients were studied. Specimens of involved and uninvolved skin from both exposed and unexposed parts of the body were compared. Changes of actinic elastosis in the papillary dermis in each patient in different stages were observed only in the exposed skin. The typical GA infiltrate was located beneath, in the upper mid-dermis. In the zone of the infiltrate in all specimens--exposed and unexposed--with no regard to the severity of actinic elastotic changes, absence or a marked reduction of elastic material was noted with engulfment of elastic remnants in giant cells. Giant cells with phagocytosis of elastotic material were mostly located on the periphery of the granuloma in the upper as well as in the deeper portion, where elastic fibers were still present. This might support the suggestion that degenerating elastic fibers promote the granulomatous reaction that leads to the formation of granuloma annulare.     

The formation of wrinkles caused by transition of keratin intermediate filaments after repetitive UVB exposure

It has been reported that the formation of wrinkles involves changes in the elastic properties of the dermis due to the denaturation of elastic fibers. Several studies have shown that the hydration condition of the stratum corneum is also important in wrinkle formation. It is, however, still unclear how the stratum corneum contributes to wrinkle formation. Here we investigated the relationship between the formation of wrinkles and changes in the physical properties and condition of the skin after repetitive ultraviolet B (UVB) irradiation of hairless mice (HR/ICR). Repetitive UVB irradiation caused wrinkles on the dorsal skin of the mice. The elasticity (E) of the stratum corneum of UVB-irradiated mice was significantly lower than that of age-matched control (unirradiated) mice. UVB exposure also caused a deterioration of the fibrous ultrastructure of keratin intermediate filaments (KIFs) in the skin. We conclude that the deterioration of KIFs in the stratum corneum caused by repetitive UVB irradiation decreases the elastic properties of the stratum corneum, resulting in the formation of wrinkles.