Aged Human Skin Research Articles

The skin as a mirror of the ageing process in the human organism – results of the ageing research in the German National Genome Research Network 2

Intrinsic human skin ageing is influenced by the individual genetic predisposition and reflects degradation processes of the body. Hormones are decisively involved in intrinsic ageing with reduced secretion of pituitary, adrenal glands, and gonads, which leads to characteristic body and skin phenotypes. A number of advances were recently made in understanding skin ageing mechanisms and major molecular changes, especiallly of the extracellular matrix, were identified. Gene expression patterns compatible with mitotic misregulation and alterations in intracellular transport and metabolism were identified in fibroblasts of ageing humans and humans with progeria. Age‐associated changes of extracellular matrix of the skin correlate well with changes been detected in the extracellular matrix of other organs of the human body. Within the National Genome Research Network 2 (NGFN‐2) in Germany, the explorative project ‘Genetic etiology of human longevity’ targets the identification of age‐related molecular pathways. For this purpose, skin models of ageing are used. Expression profiling employing cDNA microarrays from known and novel genes and RT‐PCR are employed for gene detection and confirmation. Among the potential candidate genes several interesting target genes have been identified. The evaluation of ageing‐associated genes in skin models will facilitate the understanding of global molecular ageing mechanisms in the future.

L‐Arginine supplementation or arginase inhibition augments reflex cutaneous vasodilatation in aged human skin

Full expression of reflex cutaneous vasodilatation is dependent on nitric oxide (NO) and vasodilatation is attenuated in healthy older humans. NO bioavailability in aged skin may be decreased by an age-related upregulation of arginase, which reciprocally regulates the NO-synthase (NOS) substrate L-arginine (L-Arg). We hypothesized that increased arginase activity contributes to attenuated vasodilatation in aged skin by limiting L-Arg for NOS-mediated NO synthesis. Five microdialysis fibres were placed in forearm skin of 10 young (Y, 23 +/- 1 years) and 9 older (O, 68 +/- 1 years) human subjects, serving as control (C, Ringer solution), NOS-inhibited (10.0 mM NG-nitro-L-arginine), arginase-inhibited (5.0 mM (S)-(2-boronoethyl)-L-cysteine + 5.0 mM Nomega-hydroxy-nor-L-arginine), L-arg supplemented (L-Arg; 10.0 mM L-arginine) and combined arginase-inhibited + L-Arg sites. After 20 min thermoneutral baseline, cutaneous vasodilatation was induced by passive whole-body heating to increase oral temperature (Tor) by 1.0 degrees C. Red blood cell flux was measured by laser-Doppler flowmetry over each microdialysis site. Cutaneous vascular conductance was calculated (CVC = flux/mean arterial pressure) and normalized to maximal CVC (CVCmax, 28.0 mM sodium nitroprusside + local heating to 43 degrees C). Cutaneous vasodilatation during heating was attenuated in O (Y, 42 +/- 1, versus O, 30 +/- 1%CVCmax, P < 0.001) at control sites. NOS inhibition decreased vasodilatation in both age groups compared to C (Y, 22 +/- 2; O, 18 +/- 2%CVCmax; P < 0.001). Arginase inhibition, L-Arg supplementation, and arginase inhibition + L-Arg supplementation augmented vasodilatation in O (arginase-inhibited, 46 +/- 4; L-Arg, 44 +/- 4; arginase-inhibited + L-arg, 46 +/- 5%CVCmax; P < 0.001 versus C) but not in Y (arginase-inhibited, 46 +/- 4; L-Arg, 38 +/- 4; arginase-inhibited + L-Arg, 44 +/- 4%CVCmax; P > 0.05 versus C). Increasing L-Arg for NO synthesis by either arginase inhibition or direct L-Arg supplementation restores the age-related deficit in reflex cutaneous vasodilatation.

Photoprotective and anti-skin-aging effects of eicosapentaenoic acid in human skin in vivo

Skin aging can be attributed to photoaging (extrinsic) and chronological (intrinsic) aging. Photoaging and intrinsic aging are induced by damage to human skin attributable to repeated exposure to ultraviolet (UV) irradiation and to the passage of time, respectively. In our previous report, eicosapentaenoic acid (EPA) was found to inhibit UV-induced matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts. Therefore, we investigated the effects of EPA on UV-induced skin damage and intrinsic aging by applying EPA topically to young and aged human skin, respectively. By immunohistochemical analysis and Western blotting, we found that topical application of EPA reduced UV-induced epidermal thickening and inhibited collagen decrease induced by UV light. It was also found that EPA attenuated UV-induced MMP-1 and MMP-9 expression by inhibiting UV-induced c-Jun phosphorylation, which is closely related to UV-induced activator protein-1 activation, and by inhibiting JNK and p38 activation. EPA also inhibited UV-induced cyclooxygenase-2 (COX-2) expression without altering COX-1 expression. Moreover, it was found that EPA increased collagen and elastic fibers (tropoelastin and fibrillin-1) expression by increasing transformin growth factor-beta expression in aged human skin. Together, these results demonstrate that topical EPA has potential as an anti-skin-aging agent.

Proteasomal Oscillation during Mild Heat Shock in Aging Human Skin Fibroblasts

Augmentation of proteasome machinery is emerging as a significant gerontomodulatory consequence of hormetic stimulation, such as mild heat stress. This study describes the phenomenon we term hormetic proteasomal oscillation, wherein mildly heat-stressed human fibroblasts (41 degrees C, 1 h) display an adaptation response pattern in proteasome activity. Remarkably, such response appears to be diverse in severely heat-stressed or senescent fibroblasts. This proteasomal oscillation, as an innate cellular reaction to heat and aging, however, is independent of 20S proteasome protein levels and nuclear factor-E2-related factor 2 (Nrf2) transactivation.

Expression profiling of aging in the human skin

During the last years it was shown that the aging process is controlled by specific genes in a large number of organisms ( C. elegans, Drosophila, mouse or humans). To investigate genes involved in the natural aging process of the human skin we applied cDNA microarray analysis of naturally aged human foreskin samples. For the array experiments a non-redundant set of 2135 pre-selected EST clones was used. These arrays were used to probe the patterns of gene expression in naturally aged human skin of five young (3–4 years of age) and five old (68–72 years of age) healthy persons. We found that in total 105 genes change their expression over 1.7-fold during the aging process in the human skin. Of these 43 genes were shown to be down-regulated in contrast to 62 up-regulated genes. Expression of regulated genes was confirmed by real-time PCR. These results suggest that the aging process in the human skin is connected with the deregulation of various cellular processes, like cell cycle control, cytoskeletal changes, inflammatory response, signaling and metabolism.

The effect of erythrodiol-3-acetate on the expressions of matrix metalloproteinase-1 and type-1 procollagen caused by ultraviolet irradiated cultured primary old aged human skin fibroblasts

Methanol and aqueous extracts of Styrax japonica used traditionally for the treatments of skin elastic materials were screened in vitro for the matrix metalloproteinase (MMP)-1 inhibitor actions. The methylene chloride soluble fraction of methanol extract from the stems of S. japonica showed significant MMP-1 inhibition in primary old aged human skin fibroblasts caused by ultraviolet (UV) irradiation. Main triterpenoids were isolated by repeated column chromatography. Among them, the triterpenoid erythrodiol-3-acetate reduced the expression of MMP-1 and induced the expression of type-1 procollagen at the protein levels in a dose-dependent manner caused by UV irradiated cultured old aged human skin fibroblasts. Taken together, our results suggest that erythrodiol-3-acetate plays an important role in the skin aging process caused by UV irradiation.

Influence of aging on glycosaminoglycans and small leucine-rich proteoglycans production by skin fibroblasts

Skin aging is characterised by a progressive deterioration of its functional properties, linked to alterations of dermal connective tissue. Whereas many studies have been devoted to collagen alterations during aging, the situation is less clear concerning glycosaminoglycans and proteoglycans. Particularly, the alterations of the expression of small leucine-rich proteoglycans (SLRPs), a family of proteoglycans strongly implicated in cell regulation, have never been studied. In the present study we measured glycosaminoglycans and small leucine-rich proteoglycans synthesis by skin fibroblasts from donors of 1 month to 83 years old. [3H]-glucosamine and [35S]-sulfate incorporation did not show significant differences of sulfated GAG synthesis during aging. On the other hand, a significant positive correlation was found between hyaluronan secretion and donor's age. Northern blot analysis of SLRPs mRNAs showed a significant negative correlation of lumican mRNA with donor's age, whereas decorin and biglycan mRNAs were not significantly altered. Immunohistochemical study and quantitative image analysis confirmed a decreased lumican accumulation in aged human skin. Taken together, our results suggest that impairment of glycosaminoglycans and SLRPs synthesis might be involved in the functional alterations of aged skin.

The effects of a novel synthetic retinoid, seletinoid G, on the expression of extracellular matrix proteins in aged human skin in vivo

Background Although retinoids have potential efficacy in aged skin, their side effect (skin irritation) remains a clinical problem. We designed a novel synthetic retinoid, seletinoid G, by using computer-aided molecular modeling, and investigated its effects on the expression of extracellular matrix proteins in human skin in vivo. Methods Twenty-three subjects were tested on the buttocks using 4-day occlusive application of seletinoid G and all- trans retinoic acid ( tRA). Skin irritation after topical application was quantified by the degree of erythema and cutaneous blood flow. The expression of extracellular matrix proteins and interstitial collagenase (MMP-1) in skin biopsies was investigated by immunohistochemical staining and Western blotting. Results The topical application of seletinoid G under occlusion induced no skin irritation in contrast to tRA, which caused severe erythema. The topical treatment with seletinoid G increased the expressions of type I procollagen, tropoelastin, and fibrillin-1, and reduced MMP-1 in old skin in vivo. Seletinoid G was found to inhibit not only the UV-induced decrease of type I procollagen but the UV-induced increase of MMP-1 and c-Jun protein in young skin in vivo. Conclusions Seletinoid G is a novel synthetic retinoid, which has little the side effect of skin irritation after topical application. Seletinoid G can repair altered connective tissue in old skin and inhibit UV-induced collagen deficiency in young skin.

Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts

Ultraviolet (UV) irradiation regulates UV-responsive genes, including matrix metalloproteinases (MMPs). Moreover, UV-induced MMPs cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of eicosapentaenoic acid (EPA), a dietary omega-3 fatty acid, on UV-induced MMP-1 expression in human dermal fibroblasts (HDFs). We found that UV radiation increases MMP-1 expression and that this is mediated by p44 and p42 MAP kinase (ERK) and Jun-N-terminal kinase (JNK) activation but not by p38 activation. Pretreatment of HDFs with EPA inhibited UV-induced MMP-1 expression in a dose-dependent manner and also inhibited the UV-induced activation of ERK and JNK by inhibiting ERK kinase (MEK1) and SAPK/ERK kinase 1 (SEK1) activation, respectively. Moreover, inhibition of ERK and JNK by EPA resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced activator protein-1 DNA binding activity. This inhibitory effect of EPA on MMP-1 was not mediated by an antioxidant effect. We also found that EPA inhibited 12-O-tetradecanoylphorbol-13-acetate- or tumor necrosis factor-alpha-induced MMP-1 expression in HDFs and UV-induced MMP-1 expression in HaCaT cells. In conclusion, our results demonstrate that EPA can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, EPA is a potential agent for the prevention and treatment of skin aging.

H2O2 Accumulation by Catalase Reduction Changes MAP Kinase Signaling in Aged Human Skin In Vivo

To understand the molecular alterations occurring during the aging process, we compared mitogen-activated protein (MAP) kinase activities in the intrinsically aged and photoaged skins in the same individuals. Furthermore, we investigated the molecular events related to MAP kinase changes in intrinsically aged and photoaged skins. We found that extracellular-signal-regulated kinase (ERK) activity in photoaged skin was reduced, and that the activities of c-Jun N-terminal kinase (JNK) and p38 kinase were increased compared with intrinsically aged skin in the same individuals. Phospho-c-Jun levels and activator protein 1 activities in photoaged skin were also higher than in intrinsically aged skin. Moreover, catalase activity was found to be much reduced in primary dermal fibroblasts from photoaged skin, and as a result, H2O2 accumulated more in primary dermal fibroblasts in photoaged skin. In addition, treating primary dermal fibroblasts from photoaged skin with catalase reduced H2O2 levels, reversed aging-dependent MAP kinase changes, and inhibited matrix metalloproteinase (MMP)-1 expression. Our results indicate that the accumulation of reactive oxygen species due to catalase attenuation may be a critical aspect of the MAP kinase signaling changes that may lead to skin aging and photoaging in human skin in vivo. Thus, the induction and regulation of endogenous antioxidant enzymes including catalase may offer a strategy for preventing and treating skin aging.

Topical Application of 17β-Estradiol Increases Extracellular Matrix Protein Synthesis by Stimulating TGF-β Signaling in Aged Human Skin In Vivo

To investigate the effects of topically applied 17beta-estradiol on the expression of extracellular matrix proteins in aged human skin, 17beta-estradiol (0.01%) and its vehicle (70% propylene glycol, 30% ethanol) were applied to aged (68-82 y, eight females and five males) human buttock skin under occlusion for 2 wk (three times per week). Topical 17beta-estradiol was found to increase the expression of type 1 procollagen mRNA and protein significantly in human aged skin in vivo. In addition, metalloproteinase (MMP-1 protein levels were reduced by topical 17beta-estradiol. The expressions of TGF-beta1, TGF-beta type II receptor, and Sma and Mad related (Smad)3 were increased by topical 17 beta-estradiol in aged human skin, and TGF-beta1 neutralizing antibody inhibited 17beta-estradiol-induced procollagen synthesis in cultured fibroblasts. We also found that the expressions of tropoelastin and fibrillin-1 mRNA and protein, and elastic fibers in aged skin were also increased by topical 17beta-estradiol. Topical 17beta-estradiol also increased keratinocyte proliferation and the epidermal thickness in aged human skin. We also observed the same effects of topical 17beta-estradiol in young skin. In conclusion, our results suggest that topical 17beta-estradiol treatment may improve the cutaneous function of aged human skin by improving the connective tissue and increasing epidermal thickness.

Novel Aspects of Intrinsic and Extrinsic Aging of Human Skin: Beneficial Effects of Soy Extract¶

ABSTRACTBiochemical and structural changes of dermal connective tissue substantially contribute to the phenotype of aging skin. To study connective tissue metabolism with respect to ultraviolet (UV) exposure, we performed an in vitro (human dermal fibroblasts) and an in vivo complementary DNA array study in combination with protein analysis in young and old volunteers. Several genes of the collagen metabolism such as Collagen I, III and VI as well as heat shock protein 47 and matrix metalloproteinase‐1 are expressed differentially, indicating UV‐mediated effects on collagen expression, processing and degradation. In particular, Collagen I is time and age dependently reduced after a single UV exposure in human skin in vivo. Moreover, older subjects display a lower baseline level and a shorter UV‐mediated increase in hyaluronan (HA) levels. To counteract these age‐dependent changes, cultured fibroblasts were treated with a specific soy extract. This treatment resulted in increased collagen and HA synthesis. In a placebo‐controlled in vivo study, topical application of an isoflavone‐containing emulsion significantly enhanced the number of dermal papillae per area after 2 weeks. Because the flattening of the dermal‐epidermal junction is the most reproducible structural change in aged skin, this soy extract appears to rejuvenate the structure of mature skin.

Attenuated noradrenergic sensitivity during local cooling in aged human skin

Reflex-mediated cutaneous vasoconstriction (VC) is impaired in older humans; however, it is unclear whether this blunted VC also occurs during local cooling, which mediates VC through different mechanisms. We tested the hypothesis that the sensitization of cutaneous vessels to noradrenaline (NA) during direct skin cooling seen in young skin is blunted in aged skin. In 11 young (18-30 years) and 11 older (62-76 years) men and women, skin blood flow was monitored at two forearm sites with laser Doppler (LD) flowmetry while local skin temperature was cooled and clamped at 24 degrees C. Cutaneous vascular conductance (CVC; LD flux/mean arterial pressure) was expressed as percentage change from baseline (% DeltaCVC(base)). At one site, five doses of NA (10(-10)-10(-2) m) were sequentially infused via intradermal microdialysis during cooling while the other 24 degrees C site served as control (Ringer solution + cooling). At control sites, VC due to cooling alone was similar in young versus older (-54 +/- 5 versus -56 +/- 3% DeltaCVC(base), P = 0.46). In young, NA infusions induced additional dose-dependent VC (10(-8), 10(-6), 10(-4) and 10(-2) m: -70 +/- 2, -72 +/- 3, -78 +/- 3 and -79 +/- 4% DeltaCVC(base); P < 0.05 versus control). In older subjects, further VC did not occur until the highest infused dose of NA (10(-2) m: -70 +/- 5% DeltaCVC(base); P < 0.05 versus control). When cutaneous arterioles are sensitized to NA by direct cooling, young skin exhibits the capacity to further constrict to NA in a dose-dependent manner. However, older skin does not display enhanced VC capacity until treated with saturating doses of NA, possibly due to age-associated decrements in Ca2+ availability or alpha2C-adrenoceptor function.

Histological Evaluation of a Topically Applied Retinol-Vitamin C Combination

Two double-blind studies versus vehicle were carried out to investigate the effects of a topically applied retinol plus vitamin C combination on epidermal and dermal compartments of aged or photoaged human skin. The two studies were performed on postmenopausal women who were selected for treatment based on the mild level of elastosis of their facial skin. At completion of treatment, skin biopsies were collected and processed for classical histology and immunohistochemistry. In the first study (aged skin), 8 volunteers applied the retinol- and vitamin C-containing preparation on the ventral side of one elbow and the vehicle on the other elbow twice daily for 3 months. After the 3-month treatment we observed histological changes mainly within the epidermis. The stratum corneum was thinner with a compact pattern, whereas the epidermal proliferation increased, resulting in a thickening of the viable epidermis. Moreover, the interdigitation index was increased. In the second study (photoaged skin), 11 volunteers were divided in two groups; one applied the retinol- and vitamin C-containing preparation and the other one the vehicle on their face twice daily for 6 months. Facial skin samples presented histologic hallmarks of photoaging, i.e. accumulation of elastotic material in the papillary dermis. After the 6-month topical treatment, the observed histological changes were mainly concentrated at the dermal level. Both treated and control groups showed the same distribution pattern of type I procollagen, however, the high level of type III procollagen originally observed in photoaged skin was reduced in the retinol- and vitamin C-treated group, resulting in a lower type III-to-type I procollagen ratio. Furthermore, a wide band of eosinophilic material just beneath the epidermis, devoid of oxytalan fibers and forming the ‘grenz zone’, appeared more frequently and was larger in the retinol- and vitamin C-treated group. In conclusion, our results show that repeated topical application of a preparation containing both retinol and vitamin C is able to reverse, at least in part, skin changes induced by both chronologic aging and photoaging.

Mechanisms of acetylcholine‐mediated vasodilatation in young and aged human skin

Thermoregulatory cutaneous vasodilatation (VD) is attenuated in aged skin. While acetylcholine (ACh) plays a role in thermally mediated VD, the precise mechanisms through which ACh-mediated VD acts and whether those downstream mechanisms change with ageing are unclear. We tested the hypotheses that both nitric oxide (NO)- and prostanoid-mediated pathways contribute to exogenous ACh-mediated VD, and that both are attenuated with advanced age. Twelve young (Y: 23 +/- 1 years) and 10 older (O: 69 +/- 1 years) subjects underwent infusions of 137.5 mum ACh at four intradermal microdialysis sites: control (C, Ringer solution), NO synthase inhibited (NOS-I, 10 mm l-NAME), cyclooxygenase inhibited (COX-I, 10 mm ketorolac) and NOS-I + COX-I. Red blood cell flux was monitored using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC) was calculated (laser-Doppler flux/mean arterial pressure) and normalized to maximal CVC (%CVC(max)) (28 mm sodium nitroprusside + local heating to 43 degrees C). Baseline %CVC(max) was increased in the O at COX-I sites (COX-I 16 +/- 1, NOS-I + COX-I 16 +/- 2 versus C 10 +/- 1%CVC(max); P < 0.001) but not in the young, suggesting an age-related shift toward COX vasoconstrictors contributing to basal cutaneous vasomotor tone. There was no difference in peak %CVC(max) during ACh infusion between age groups, and the response was unchanged by NOS-I (O: NOS-I 35 +/- 5 versus C 38 +/- 5%CVC(max); P = 0.84) (Y: NOS-I 41 +/- 4 versus C 39 +/- 4%CVC(max); P = 0.67). COX-I and NOS-I + COX-I attenuated the peak CVC response to ACh in both groups (COX-I O: 29 +/- 3, Y: 22 +/- 2%CVC(max) versus C; P < 0.001 both groups; NOS-I + COX-I O: 32 +/- 3 versus Y: 29 +/- 2%CVC(max); versus C; P < 0.001 both groups). ACh mediates cutaneous VD through prostanoid and non-NO-, non-prostanoid-dependent pathways. Further, older subjects have a diminished prostanoid contribution to ACh-mediated VD.

Increased expression of 14-3-3ɛ protein in intrinsically aged and photoaged human skin in vivo

Skin aging is a complicated process associated with the passage of time and environmental exposure, especially to UV light. This aging phenomenon is related to alterations in various cellular mechanisms, such as changes in apoptosis, perturbations to cellular signaling, and an increased genetic instability. In this study, we investigated changes of proteins involved in intrinsic aging by the proteomic analysis of human sun-protected (upper inner arm) young and aged dermis. One of the proteins upregulated in aged dermis was identified as 14-3-3ɛ. This protein is an isoform of 14-3-3 protein, which is involved in cellular processes like signal transduction, cell cycle arrest, and apoptosis. 14-3-3ɛ is consistently found to be upregulated in the sun-protected dermis of aged skin, by Western blotting and immunohistochemical staining. In addition, we demonstrate that the expression of 14-3-3ɛ is further upregulated in the sun-exposed (photodamaged) dermis, and that the UV irradiation of young skin significantly upregulates 14-3-3ɛ in vivo. Our results suggest the possibility that the cellular processes related to 14-3-3ɛ protein play an important role in the photoaging and intrinsic aging of human skin.

Novel Aspects of Intrinsic and Extrinsic Aging of Human Skin: Beneficial Effects of Soy Extract¶

Biochemical and structural changes of the dermal connective tissue substantially contribute to the phenotype of aging skin. To study connective tissue metabolism with respect to ultraviolet (UV) exposure, we performed an in vitro (human dermal fibroblasts) and an in vivo complementary DNA array study in combination with protein analysis in young and old volunteers. Several genes of the collagen metabolism such as Collagen I, III and VI as well as heat shock protein 47 and matrix metalloproteinase-1 are expressed differentially, indicating UV-mediated effects on collagen expression, processing and degradation. In particular, Collagen I is time and age dependently reduced after a single UV exposure in human skin in vivo. Moreover, older subjects display a lower baseline level and a shorter UV-mediated increase in hyaluronan (HA) levels. To counteract these age-dependent changes, cultured fibroblasts were treated with a specific soy extract. This treatment resulted in increased collagen and HA synthesis. In a placebo-controlled in vivo study, topical application of an isoflavone-containing emulsion significantly enhanced the number of dermal papillae per area after 2 weeks. Because the flattening of the dermal-epidermal junction is the most reproducible structural change in aged skin, this soy extract appears to rejuvenate the structure of mature skin.

Regulation Effect of 2',4',7-Trihydroxyisoflavone on the Expression of Matrix Metalloproteinase-1, 2 in Ultraviolet-B Irradiated Primary Cultured Old Aged Human Skin Fibroblasts

Long term and repeated exposure of UV light on the skin often induces chronic skin diseases such as skin cancer as well as photoaging, and the mechanisms of these skin damages are closely associated with up-regulation of matrix metalloproteinase's (MMPs) activities. We investigated the effect of 2',4',7-trihydroxyisoflavone purified from the whole plants of Viola hondoensis W. BECKER et H BOISSIEU (Violaceae) on the expression of MMPs in UV-B irradiated old aged human skin fibroblasts. 2',4',7-trihydroxyisoflavone markedly reduced UV-induced MMP-1 expression, but not MMP-2, at the both mRNA and protein levels in a dose-dependent manner. Our report is the first description for the ability of 2',4',7-trihydroxyisoflavone to regulate MMP-1 expression from ultraviolet-B irradiated primary cultured old aged human skin fibroblasts.

Alteration of the TGF-β/SMAD pathway in intrinsically and UV-induced skin aging

In an effort to characterize transforming growth factor (TGF-β) signaling and to determine its association with the aging and photoaging processes, we directly compared the expressions of TGF-β/SMAD in intrinsically aged and photoaged human skin in vivo. By using an RNase protection assay and by immunohistochemistry, we found that the expression levels of TβRII mRNA and protein in the epidermis of the forearm (sun-exposed) of the elderly were significantly lower than that of the upper-inner arm (sun-protected) skin of the same individual. In the epidermis, the expressions of Smad7 mRNA in both the intrinsically aged and photoaged skin of the elderly were higher than in the sun-protected skin of the young, and this was elevated in the photoaged epidermis. Decreased pSmad2 immunoreactivity was observed in the epidermis of photoaged forearm skin versus matched intrinsically aged skin. This decrease was also found in the epidermis of upper-inner arm skin of the elderly versus the young. These results suggest that the UV-induced down-regulation of TβRII and the concerted over-expression of Smad7 may trigger the inhibition of the TGF-β-induced phosphorylation of Smad2.

Expression of psoriasis-associated fatty acid-binding protein in senescent human dermal microvascular endothelial cells.

Aging is associated with the progressive pathophysiologic modification of endothelial cells. In vitro endothelial cell senescence is accompanied by proliferative activity failure and by perturbations in gene and protein expressions. Moreover, this cellular senescence in culture has been proposed to reflect processes that occur in aging organisms. In order to observe the changing patterns of protein expression in senescent human dermal microvascular endothelial cells (HDMECs), proteins obtained from both early- and late-passaged HDMECs were separated by two-dimensional electrophoresis, visualized by silver staining, and quantified by image processing. Proteins of interest were extracted by in-gel digestion with trypsin and quantified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), by searching the National Center for Biotechnology Information protein-sequence database. More than 2000 spots were detected by 2D electrophoresis within a linear pH range of 3-10. Twenty-two major differentially expressed spots were observed in serially passaged HDMECs and identified with high confidence by MALDI-TOF-MS. One of these spots was found to be a 14-15 kDa psoriasis-associated fatty acid-binding protein (PA-FABP) with high affinity for long-chain fatty acids. The expression of PA-FABP was confirmed to be elevated in senescent HDMECs (passage 20) by fluorescence-activated cell sorting (FACS), confocal laser microscopy, and by immunohistochemistry in aged human skin tissue. Our results suggest that the overexpression of FABP in cultured senescent HDMECs is closely related to skin aging.