Epidermal innervation correlates with severity of photodamage

Abstract Intraepidermal nerve fibers were studied by electron microscopy in chronically photodamaged preauricular skin and in paired sun-protected postauricular sites of 20 Caucasian women aged 56–70 years. As previously reported, basal keratinocytes in the sun-exposed skin showed various degrees of degenerative changes including intracellular vacuolar structures and widened intercellular spaces. Neurites were frequently closely apposed to basal keratinocytes in preauricular sun-exposed skin, but were observed less than 10% as often in sun-protected postauricular skin. When degree of epidermal photodamage was quantified by means of the number of degenerated keratinocytes per 100 keratinocytes in the basal layer, the number of intraepidermal nerve libers was significantly correlated by linear regression analysis to the severity of epidermal photodamage (r=0.913) independent of anatomical sites. These results demonstrate for the first time a correlation between degree of epidermal innervation and chronic photodamage and suggest the possibility of neural involvement in the pathophysiology and/or repair of photodamaged skin.     

Photoageing shows histological features of chronic skin inflammation without clinical and molecular abnormalities

BACKGROUND: Photodamage is characterized by degradation of collagen and accumulation of abnormal elastin in the superficial dermis. Mast cells and macrophages, which are found in higher numbers in photoaged skin, have been implicated in this process. OBJECTIVES: To analyse the phenotype of haematopoietic-derived infiltrating cells in photodamaged skin. METHODS: Chronically sun-exposed (preauricular) and control sun-protected (postauricular) skin was recovered from eight healthy subjects undergoing cosmetic surgery (facial lifting). RESULTS: Histological analysis showed that sun-exposed skin harboured more infiltrating mononuclear cells than sun-protected skin. Cellular infiltrates were found at the periphery of areas of elastolysis around hair follicles in sun-exposed sites, whereas they were found in the interfollicular dermis around blood vessels and around hair follicles in sun-protected samples. Immunohistochemical analysis revealed an increased number of mast cells, macrophages and CD4+ CD45RO+ T cells in sun-exposed dermis as well as a higher number of CD1a+ dendritic cells in sun-exposed epidermis, compared with the sun-protected samples. Thus photoageing displays histological features of chronic skin inflammation. However, no molecular sign of inflammation was observed and we even found a decreased expression of interleukin-1beta mRNA in sun-exposed compared with sun-protected sites. Furthermore, the patients' skin looked normal and did not display any clinical inflammation. CONCLUSIONS: Collectively, these data show that chronic ultraviolet irradiation induces alterations of innate immune cells which are recruited in sun-exposed skin without being activated.     

GLGI技术初步验证系统性红斑狼疮患者CD4+、CD8+T细胞基因表达谱

目的 研究GLGI技术鉴定系统性红斑狼疮患者LongSAGE标签的方法。方法 运用GLGI技术,将包括17 bp的SAGE标签及单一碱基和oligo(dT)锚定引物进行PCR扩增,得到表达基因的3'端序列,并确认表达基因。结果 单一匹配标签12个有9个获得有效扩增,包括原来的17 bp的SAGE标签序列,其长片段测序后有>85%的碱基与已知基因相符合,表明为该已知基因。20个无匹配标签中未发现有新的基因。结论 GLGI技术能够有效识别基因,LongSAGE-GLGI技术是鉴定SAGE大规模标签库较好的方法。     

Telomere length of the skin in association with chronological aging and photoaging

BACKGROUND: Telomere shortening has been implicated in cellular senescence, which may cause certain aging phenotypes. OBJECTIVE: To reveal whether telomere shortening is associated with chronological aging and/or photoaging of the skin, we measured telomere length in the epidermis and in the dermis from sun-protected and from sun-exposed sites of the skin. METHODS: Seventy-six specimens of epidermis from sun-protected sites and 24 specimens of epidermis from sun-exposed sites were analyzed. Sixty specimens of the dermis were also analyzed. In six cases, epidermal specimens from sun-protected and from sun-exposed sites of the same individual were analyzed. RESULTS: Comparison of telomere lengths revealed that the epidermis has shorter telomeres than the dermis. Telomere length in the epidermis and in the dermis was reduced with age, and average telomere shortening rates in the epidermis and in the dermis were 9 and 11 bp/yr, respectively. Unexpectedly, telomere length was not significantly different between epidermis from sun-exposed sites and from sun-protected sites. CONCLUSION: We could not show the evidence that telomere shortening is associated with photoaging of the skin.     

Photoaging-associated changes in epidermal proliferative cell fractions in vivo

The epidermis is a dynamic epithelium with constant renewal throughout life. Epidermal homeostasis depends on two types of proliferative cells, keratinocyte stem cells (KSCs), and transit amplifying (TA) cells. In the case of chronologic aging, levels of KSCs tend to decrease and change functionally. However, little is known about the effect of photoaging on epidermal proliferative subtype populations. The aim of this study was to validate involucrin/β1-integrin ratio as a molecular marker of epidermal photoaging, and to investigate the effects of photoaging caused by chronic UV exposure on the proliferative subtype populations. A total of 15 male volunteers (age range 20–24 and 77–85 years, Fitzpatrick skin phototype III–IV) provided sun-exposed and sun-protected skin samples for real-time RT-PCR, Western blot analysis and immunostaining. Fractional changes in proliferative subtype populations in photoaged and chronologically aged skins were analyzed by flow cytometry. The expression of β1-integrin was found to be significantly reduced in photoaged skin and ratios of the expressions of involucrin to β1-integrin were increased 2.6-fold only in elderly subjects. Interestingly, immunostaining of the sun-exposed skins of elderly subjects showed aberrant β1-integrin expression over the basal layer and greater numbers of Ki-67-positive cells than in sun-protected buttock skin. Flow cytometric analysis revealed that the proportion of KSCs to TA cells was reversed in sun-exposed and sun-protected skins of elderly subjects. Our results suggest that KSC numbers may be lower in photoaged skin than in chronologically aged skin and could be applied to hyperplastic pattern of photoaging. These findings suggest that the epidermis of photoaged skin is impaired in terms of its proliferative potential by attempting to repair chronic UV exposure and that photoaging may be associated with alteration in the two proliferative cell fractions.     

Chronic sun exposure alters both the content and distribution of dermal glycosaminoglycans

Summary Chronic sun exposure leads to structural and functional alterations in exposed skin. Photoageing is a process distinct from the changes taking place due to chronological ageing. Unique alterations in the dermal extracellular matrix occur as a result of photoageing and are responsible for many of these physiological changes taking place in sun-damaged skin. Accompanying the deposition of abnormal elastic tissue, or solar elastosis, are significant alterations in dermal glycosaminoglycans (GAGs). Accumulation of GAGs as a result of photoageing as demonstrated in both humans and animal models of photoageing seems almost paradoxical in view of the large amounts of GAGs present in the skin of newborns, making their skin well hydrated and supple, in sharp contrast to the weathered appearance of photoaged skin. We investigate the relative GAG content of photoaged skin using immunoperoxidase stains specific for hyaluronic acid and chondroitin sulphate, and determine the location of these GAGs using confocal laser scanning microscopy. Our results demonstrate significant increases in GAG staining in sun-damaged vs. sun-protected skin from the same individuals, as measured by computer-based image analysis. Furthermore, confocal laser scanning microscopy reveals that the increased dermal GAGs in sun-damaged skin are deposited on the elastotic material of the superficial dermis of photodamaged skin, and not between collagen and elastic fibres as in normal skin. The abnormal location of GAGs on these fibres may explain the apparent paradoxical weathered appearance of photodamaged skin despite increased GAGs.     

Activation of heparanase by ultraviolet B irradiation leads to functional loss of basement membrane at the dermal–epidermal junction in human skin

Recently, we reported that heparanase plays important roles in barrier-disrupted skin, leading to increased interaction of growth factors between epidermis and dermis and facilitating various cutaneous changes, including epidermal hyperplasia and wrinkle formation. However, the role of heparanase in sun-exposed skin remains unknown. Here, we show that heparanase in human keratinocytes is activated by ultraviolet B (UVB) exposure and that heparan sulfate of perlecan is markedly degraded in UVB-irradiated human skin. The degradation of heparan sulfate resulted in a marked reduction of binding activity of the basement membrane for vascular endothelial growth factor, fibroblast growth factor-2 and -7 at the dermal–epidermal junction. Degradation of heparan sulfate was observed not only in acutely UVB-irradiated skin, but also in skin chronically exposed to sun. Interestingly, heparan sulfate was found to be degraded in sun-exposed skin, but not in sun-protected skin. These findings suggest that chronic UVB exposure activates heparanase, leading to degradation of heparan sulfate in the basement membrane and increased growth factor interaction between epidermis and dermis. These changes may facilitate photo-aging.     

高通量GLGI鉴定及分析白念珠菌LongSAGE标签

目的鉴定并初步分析白念珠菌长片段基因表达序列分析(LongSAGE)标签。方法建立了一种高通量鉴定LongSAGE标签的技术,应用该技术可批量扩增白念珠菌LongSAGE标签,使其由17bp扩增至相应的3'cDNA末端,扩增产物TA克隆后进行测序及序列分析(GLGI)。结果30条多重匹配标签中28条得到稳定有效的扩增,40个无匹配标签中30条得到有效扩增,其中21条证实为新表达序列标签(EST),可能代表新的基因,9条和已知基因有一定同源性,可能是已知基因或其同一家族基因。结论应用高通量GLGI技术成功鉴定了白念珠菌LongSAGE标签,为进一步研究白念珠菌菌相转换机制奠定了基础。     

Culture and drug biotransformation capacity of adult human keratinocytes from post-mortem skin

Summary 'I'hc aim of this study was to analyse viability, growth, differentiation and drug metabolic capacity of cultured human keratinocytes obtained from post-mortem skin. Epidermal cells were prepared from 1-day post-mortem paired sun-exposed (outer) and sun-protected (inner) sites of the upper arm. of donors aged 47–80 years, The percentage of viable cells obtained from post-mortem skin was only slightly lower than that usually obtained for keratinocytes isolated from fresh skin, and no alterations of epidermal markers were noted. Keratinocytes isolated post-mortem from non-exposed skin had a higher viability (78 versus 73%). and a more active proliferation, while their attachment rate, keratin composition, lipid synthesis capacity and transglutaminase activity levels were similar to those of epidermal cells obtained from the sun-exposed skin. Keratinocytes Isolated from postmortem skin expressed various phase I and II activities at levels similar to those obtained with keratinocytes isolated from fresh skin while drug metabolizing enzyme activities were consistently higher in sun-exposed compared to sun-protected cells. The results support the conclusion that skin collected post-mortem can represent an alternative source of viable and functional epidermal cells, and that the functional changes that occur in adult keratinocytes habitually exposed to the sun, affect much more strongly the drug metabolism capacity than the expression of differentiation markers.     

Effect of age and anatomical site on density of sensory innervation in human epidermis

BACKGROUND: Aging leads to decline of multiple cutaneous physiological functions including decreased sweating, immune responsiveness, thermoregulation, DNA repair, and sensory and tactile perception. Interestingly, sensory perception, like that for pain or spatial acuity, varies in different body parts. OBJECTIVE: To evaluate epidermal innervation according to age and anatomical site. METHODS: Eighty-two biopsy samples from surgical procedures involving 82 patients of different ages (20-93 years) were analyzed. Four anatomical sites were examined: 2 from facial areas (upper eyelid and preauricular area) and 2 from truncal areas (abdomen and mammary area). Epidermal innervation was detected using a marker of neural cells, the protein gene product 9.5. The basement membrane was stained with type IV collagen antibodies. The epidermal area occupied by nerve endings was then calculated using image analysis. RESULTS: A trend displaying age-associated decreased epidermal innervation of facial skin was found. Epidermal innervation of abdominal skin did not change with age, and an age-associated increased innervation was observed in mammary skin. Also, the number of epidermal nerves in facial areas tested (palpebral and preauricular areas) was significantly higher than their number in the abdomen and mammary area. Eyelid epidermis showed the highest ratio of nerve fiber surface to epidermal surface. CONCLUSIONS: Epidermal nerve density variations could explain the different sensitivity threshold in different parts of the body. Decreased spatial discrimination with aging may be associated with decreased epidermal nerve density.     

Ultrastructural characterization of microvasculature in photoaging

The cutaneous microvasculature was examined by electron microscopy in order to compare its characteristics in photodamaged preauricular skin and in sun-protected postauricular sites of 15 Japanese women aged 58-81 years. The characteristic ultrastructural features of the microvasculature in photodamaged skin compared with those in sun-protected skin included dilated vessels embedded in elastin which depressed endothelial cells, vessels surrounded by a thick amorphous material composed of multiple laminations of a basement membrane-like material, and activated endothelial cells which had increased numbers of cytoplasmic organelles and pinocytotic vesicles. A novel finding of this study in photodamaged vessels was an increased formation of new vessels (angiogenesis) via two distinct pathways. In severe elastosis, activated endothelial cells with densely packed intracytoplasmic microfilaments extended large pseudopods into the elastotic material. In contrast, isolated mesenchymal cells, which possessed immature Weibel-Palade bodies, were scattered around pre-existing vessels within the Grenz zone. In some cases, many mesenchymal cells with electron-lucent cytoplasms aggregated and interconnected by cytoplasmic processes, which was followed by the formation of vascular structures. These results suggest that there are significant ultrastructural differences in vessels between photoaged and intrinsically aged facial skin and that the photodamaged microvascular system is characterized by the co-existence of regressive changes and angiogenesis.     

Expression of e-cadherin and beta-catenin in cutaneous squamous cell carcinoma and its precursors

The E-cadherin-beta-catenin complex regulates the architectural integrity of epithelia by mediating intercellular adhesion. Down-regulation of its expression may contribute to invasion and metastatic behavior of carcinoma cells. Several studies demonstrated an abnormal expression of E-cadherin, beta-catenin, or both in various carcinomas, including non-melanoma skin cancer. The aim of the present study was to investigate the involvement of E-cadherin-catenin adhesion system in the progression of human cutaneous squamous cell carcinoma (SCC). For that purpose, sections from normal skin, skin showing solar elastosis (SE), solar keratosis (SK), and SCC were stained with monoclonal antibodies against E-cadherin and beta-catenin. Evaluation of the staining results was performed using a semi-quantitative method in which pattern and intensity of staining, percentage of positive cells, and cytoplasmic staining were evaluated. Normal skin and skin showing mild and moderate solar elastosis strongly expressed membranous E-cadherin and beta-catenin. E-cadherin expression was progressively reduced in the epidermis of skin with severe solar elastosis through solar keratosis to SCC. The same phenomenon was observed for beta-catenin starting from solar keratosis. In some cases of SCC, additional cytoplasmic staining was observed. We found no correlation between E-cadherin and beta-catenin expression and tumor differentiation or between SCC from sun-exposed and sun-protected skin. Statistical analysis revealed correlation between expression of both E-cadherin and beta-catenin and the morphology of the lesion. These results support a gradual evolution from severely sun-damaged skin to SCC, not only on a morphologic level, but also at the molecular level.     

Topographic variations in normal skin, as viewed by in vivo reflectance confocal microscopy.

Near-infrared confocal microscopy is a new tool that provides skin images in vivo, with high resolution and contrast at a specific depth. Regional variations in live human skin viewed by confocal microscope have not been studied so far. In vivo reflectance confocal microscopy was performed in 10 adults (eight males, two females) of various skin phototypes. Six topographic sites were studied in each subject: forehead, cheek, inner and outer forearm surfaces, lower back and leg. Epidermal thickness at suprapapillary epidermal plates and rete pegs was measured during real-time imaging and the number and diameter of epidermal keratinocytes in each epidermal cell layer as well as the characteristics of dermal papillae were defined from the grabbed images. Stratum corneum appeared brighter in sun-exposed than in sun-protected areas and particularly pronounced in heavily pigmented individuals. The epidermal thickness at rete pegs, but not the suprapapillary epidermal plate, was greater in sun-exposed areas than in sun-protected sites except forearm flexor surface. The en face numerical density of granular keratinocytes is greater on the face as compared with all other sites, whereas the surface density of spinous keratinocytes is greater on sun-protected sites. Additionally, the number of basal keratinocytes per millimeter length of dermoepidermal junction is greater in sun exposed areas. Interestingly, the dermal papillae shape varies and their sizes increase in circumference from sun-exposed to sun-protected sites, as observed at a specific depth below the stratum corneum. In summary, our results demonstrate that near infra-red reflectance confocal microscopy is a feasible tool for microscopic analysis of skin morphometry in vivo.     

Effect of aging and habitual sun exposure on the genetic response of cultured human keratinocytes to solar-simulated irradiation.

Aging and chronic sun exposure are known to be associated with decreased cutaneous immune function, changes in the balance between epidermal proliferation and differentiation, and a greatly enhanced risk of photocarcinogenesis. However, their specific effects on the response of human keratinocytes to ultraviolet (UV) irradiation are unknown. We therefore asked whether aging and photoaging modulate the response at the mRNA level to UV-inducible genes implicated in immunomodulation and/or growth control. Cultured human keratinocytes derived from newborn, young adult, and old adult donors were exposed to a single physiologic dose of solar-simulated UV or sham irradiation and harvested at 1, 4, 8, 24, and 48 h post-irradiation for northern blot analysis. Specific mRNA was detected using cDNA probes encoding the proto-oncogenes c-fos and c-myc and the growth-arrest and DNA damage (GADD153) gene, all recently shown by our laboratory to be modulated by UV in newborn keratinocytes; interleukin (IL)-1a, IL-1b, and the IL-1 receptor antagonist (IL-1ra), two keratinocyte cytokines and their competitive inhibitor, implicated in the immunomodulatory effect of UV; and SPR2, a recently cloned gene known to be induced during normal keratinocyte differentiation and by lethal UV-C irradiation. Our data suggest that aging alone strikingly increases the baseline expression of SPR2 and IL-1ra but has relatively little effect on the response to UV for the other genes examined. In contrast, the combination of aging and habitual sun exposure, so-called photoaging, markedly increases c-fos inducibility and decreases baseline expression of SPR2 and IL-1ra relative to that in cells from sun-protected skin of the same donors. The implied alterations in signal transduction and differentiation state observed in cells derived from habitually sun-exposed sites of old adults may explain in part the predisposition to photocarcinogenesis in photoaged skin.     

The use of a 3895 bp mitochondrial DNA deletion as a marker for sunlight exposure in human skin

Previous work has examined the use of mitochondrial DNA (mtDNA) damage as a biomarker of cumulative sun exposure in human skin. These studies have simply compared mtDNA damage between sun-protected and sun exposed skin. This approach is limited because non-melanoma skin cancer (NMSC) is predominantly formed on body sites which are 'usually' sun exposed as opposed to sites which are 'occasionally' sun exposed and as such they differ in their cumulative ultraviolet (UV) exposure. This study addresses this limitation by investigating the frequency of occurrence of a rarely reported 3895 bp mtDNA deletion in 104 age-matched human skin samples taken from different sun-exposed body sites. There was a significant increase in the deletion frequency with increasing UV exposure (p<0.0001). Furthermore there was a significantly greater deletion frequency in 'usually' sun exposed compared with 'occasionally' sun-exposed body sites in both the dermis (p=0.0018) and epidermis (p<0.0001). Investigation of the 3895 bp deletion in the same NMSC samples used in a previous study of the 4977 bp common deletion, showed a greater frequency of the 3895 bp deletion (8/10 vs 4/10, respectively). Additionally, we have linked the 3895 bp deletion with the UVR component of sunlight by inducing the deletion in vitro with repetitive sub-lethal doses of a UVA+UVB light source.     

表皮p63和CD44与年龄及曝光部位的相关性

目的 探讨p63与CD44在皮肤衰老过程中与年龄、紫外线的关系。方法 采用免疫组化方法检测121例正常全厚皮肤标本中p63与CD44的表达,光镜下观察并计算表皮基底层p63阳性细胞百分比,用图像分析软件image proplus 6.0测量表皮层p63与CD44表达的吸光度值。结果 ①表皮基底层p63阳性细胞百分比与年龄呈负相关（r = -0.218,P < 0.05）,51 ~ 79岁组最低;但在曝光部位、半曝光部位与非曝光部位间的差异无统计学意义。②表皮p63的表达强度与年龄在曝光部位呈负相关（r = -0.389,P = 0.010）,在半曝光部位无相关性,在非曝光部位呈正相关（r = 0.341,P < 0.05）;三个部位表达强度的差异在10 ~ 30岁组无统计学意义,在31 ~ 50岁组与51 ~ 79岁组由强至弱依次为非曝光部位、半曝光部位、曝光部位。③表皮CD44的表达强度与年龄之间呈微弱负相关（r = -0.083,P < 0.05）,10 ~ 30岁组最高;但在曝光部位、半曝光部位与非曝光部位间的差异无统计学意义。结论 表皮基底层p63阳性细胞百分比、表皮CD44的表达强度和年龄呈负相关,和紫外线照射量无关。表皮p63表达强度在31 ~ 50岁组与51 ~ 79岁组的表达强度依曝光多少而递减。     

Real‐time polymerase chain reaction analysis of a 3895‐bp mitochondrial DNA deletion in epithelial swabs and its use as a quantitative marker for sunlight exposure in human skin

Background The use of mitochondrial DNA (mtDNA) damage as a reliable and highly sensitive biomarker of ultraviolet (UV) radiation exposure in both the dermis and epidermis has now been well developed by our group and others. We have previously identified a 3895‐bp mtDNA deletion which occurred more frequently and to a higher level in usually sun‐exposed skin as opposed to occasionally sun‐exposed skin. This work focused on older‐aged individuals and, in particular, perilesional, histologically normal skin biopsies taken from patients with skin cancer. Objectives To develop novel, less‐invasive methods of obtaining skin samples (i.e. epidermis) from volunteers covering a much wider age range and larger number of individuals (n = 239). Methods The 3895‐bp deletion was quantified by a specific real‐time polymerase chain reaction assay in normal human epidermis samples taken from three body sites with differing sun exposure. Results The results show a statistical increase of the level of the 3895‐bp deletion with increasing sun exposure in the epidermal swabs of human skin (P < 0·001) and with increasing age of the donor in the needle biopsy samples. Conclusions These data suggest that the upper layers of the epidermis are an accessible and reliable site for assessing mtDNA damage caused by UV exposure.