Shigeru Moriwaki

The Role of Elastases Secreted by Fibroblasts in Wrinkle Formation: Implication Through Selective Inhibition of Elastase Activity ¶

Abstract We have previously demonstrated that decreases in skin elasticity, accompanied by increases in the tortuosity of elastic fibers, are important early events in wrinkle formation. In order to study the role of elastases in the degeneration of elastic fibers during wrinkle formation we examined the effects of an inhibitor of skin fibroblast elastase, N-phenethylphosphonyl-l-leucyl-l-tryptophane (NPLT), on wrinkle formation in hairless mice skin following UV irradiation. Dorsal skins of hairless mice were exposed daily to UV light for 18 weeks at doses of 65–95 mJ/cm2 and treated topically with 100 μL of 1 mM NPLT immediately after each UV irradiation. Wrinkles on dorsal skins were evaluated from week 6 through week 18. The daily exposure of mouse skin to UV light with less than 1 minimal erythemal dose significantly enhanced the activity of elastase in the exposed skin by week 4, and the elevated levels of elastase activity were significantly reduced by the in vitro incubation with NPLT in a dose-dependent manner to a level similar to that in unexposed mice skin, indicating that NPLT can efficiently inhibit the UV-inducible elastase activity. Topical application of NPLT significantly suppressed wrinkle formation when compared with vehicle controls by week 15 of treatment (P < 0.05). Histochemistry of elastic fibers with Orcein staining demonstrated that there were no obvious decreases of the fine elastic fibers in UV-exposed NPLT-treated skin in contrast to their marked decreases in the UV-exposed vehicle-treated skin. These findings suggest that skin fibroblast elastase plays a decisive role in wrinkle formation through the degeneration of elastic fiber.

Inhibition of ultraviolet‐B‐induced wrinkle formation by an elastase‐inhibiting herbal extract: implication for the mechanism underlying elastase‐associated wrinkles

Background  Previously, we have demonstrated that fibroblast‐derived elastase plays an essential role in the increased three‐dimensional tortuosity of elastic fibers, contributing to the loss of skin elasticity in UV‐B‐exposed skin. This decrease in skin elasticity is closely associated with the formation of wrinkles induced by UV exposure.

Dermal fluid translocation is an important determinant of the diurnal variation in human skin thickness.

Background Ultrasonography has been used as a non‐invasive approach to measure skin thickness. To date there have been no studies on diurnal variations in skin thickness.

Ovariectomy is sufficient to accelerate spontaneous skin ageing and to stimulate ultraviolet irradiation‐induced photoageing of murine skin

Summary Background  Wrinkling and sagging of the skin during photoageing is physiologically associated with diminished elasticity, which can be attributed to increased fibroblast‐derived elastase activity. This degrades the dermal elastic fibres needed to maintain the three‐dimensional structure of the skin. We previously reported that ovariectomy accelerates ultraviolet (UV)B‐induced wrinkle formation in rat hind limb skin by altering the three‐dimensional structure of elastic fibres.

Neprilysin Is Identical to Skin Fibroblast Elastase: ITS ROLE IN SKIN AGING AND UV RESPONSES

Although human skin fibroblast (HSF) elastase has been characterized as a membrane-bound metalloproteinase, little is known about its structure, amino acid sequence, and encoding gene. As there are similarities in the molecular weights and inhibitory profiles of HSF elastase and neprilysin (neutral endopeptidase 24.11 (NEP)), in this study we tested the hypothesis that they are identical using immunoprecipitation and transfection methods. An immunoprecipitation study demonstrated that HSF elastase activity co-immunoprecipitated with anti-NEP in lysates of cultured HSF. Transfection of an NEP cDNA expression vector into COS-1 cells elicited the expression of HSF elastase and NEP activities in the transfected cells. These findings strongly suggest that HSF elastase is identical to NEP, which functions mainly in neuron-associated cells to degrade neuropeptides. Analysis of the expression pattern of NEP revealed that its expression was remarkably up-regulated at the gene, protein, and enzymatic activity levels during the replicative senescence of cultured HSF. Further, the activity of NEP was markedly enhanced in a pattern similar to elastase activity during the intrinsic aging of mouse skin, in UVA-exposed HSF as well as in HSF treated with conditioned medium from UVB-exposed human keratinocytes. Analysis of the cytokine profile for the stimulation of NEP and HSF elastase activities in HSF demonstrated that among the 11 cytokines tested, IL-1α, IL-1β, IL-6, IL-8, and GM-CSF had the potential to significantly stimulate both activities similarly, again supporting the identity of HSF elastase and NEP.

Depigmentation caused by application of the active brightening material, rhododendrol, is related to tyrosinase activity at a certain threshold.

BACKGROUND Tyrosinase, the rate-limiting enzyme required for melanin production, has been targeted to develop active brightening/lightening materials for skin products. Unexpected depigmentation of the skin characterized with the diverse symptoms was reported in some subjects who used a tyrosinase-competitive inhibiting quasi-drug, rhododendrol. OBJECTIVE To investigate the mechanism underlying the depigmentation caused by rhododendrol-containing cosmetics, this study was performed. METHODS The mechanism above was examined using more than dozen of melanocytes derived from donors of different ethnic backgrounds. The RNAi technology was utilized to confirm the effect of tyrosinase to induce the cytotoxicity of rhododendrol and liquid chromatography-tandem mass spectrometry was introduced to detect rhododendrol and its metabolites in the presence of tyrosinase. RESULTS Melanocyte damage was related to tyrosinase activity at a certain threshold. Treatment with a tyrosinase-specific siRNA was shown to dramatically rescue the rhododendrol-induced melanocyte impairment. Hydroxyl-rhododendrol was detected only in melanocytes with higher tyrosinase activity. When an equivalent amount of hydroxyl-rhododendrol was administered, cell viability was almost equally suppressed even in melanocytes with lower tyrosinase activity. CONCLUSION The generation of a tyrosinase-catalyzed hydroxyl-metabolite is one of the causes for the diminishment of the melanocyte viability by rhododendrol.

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.

Age-Related Alterations of Echogenicity in Japanese Skin

Background: Ultrasonic methods are commonly used for the noninvasive measurement of skin thickness. The effects of ageing on the skin have been reported to differ between Asians and Caucasians. Therefore, it is possible that the echogenicity of the skin on ultrasonographic images reflects differences in the skin architecture and properties that may vary among races and environments. Objective and Methods: Since there have been a few reports on ageing-related changes in ultrasonic echogenicity in Caucasian skin, but not in Asian skin, we evaluated age-related changes in the echogenicity of ultrasonographic images by the ultrasonic B mode at 3 sites on the face (forehead, eye corners, cheeks) considered as sun-exposed areas and on the ventral forearms considered as weakly sun-exposed areas in 130 Japanese females aged from 18 to 83 years. The dermis was divided into 3 layers (upper, middle, lower), and echogenicity was evaluated in each area. Results: No age-related change in echogenicity was observed in the entire dermis. When the dermis was divided into 3 layers (upper, middle and lower dermis), the echogenicity was decreased in the upper layer and increased in the lower layer with age in all skin areas tested. In contrast, there were no age-related changes of echogenicity in the middle dermis. Conclusion: These findings suggest that, as for Caucasians, echogenicity in the upper and lower dermis is a useful tool for evaluating skin ageing in the Japanese.

Ovariectomy Accelerates Photoaging of Rat Skin

We have previously reported the formation of wrinkles, a decrease in skin elasticity and a loss in the linearity of dermal elastic fibers in rat hind limb skin irradiated with ultraviolet radiation in wavelength ranging 290–320 nm (UVB) at a suberythemal dose for 6 weeks. Estrogens are considered effective in preventing photoaging in postmenopausal females, but the role of estrogen in the skin remains unclear. In this study we have evaluated the influence of short‐term chronic UVB irradiation at a suberythemal dose on the skin of ovariectomized rats. An ovariectomy or a sham operation was performed on each 3 week–old female Sprague‐Dawley rat. Starting 1 week after the operation the hind limb skin of each rat was irradiated with UVB at a suberythemal dose (130 mJ/cm2) three times a week for 3 or 6 weeks. Decreases in elasticity and wrinkle formation in the skins of ovariectomized animals were induced more quickly than in the skins of sham‐operated animals following UVB irradiation. The linearity of elastic fibers in the ovariectomy group decreased significantly compared with the sham‐operation group, but erythema in the ovariectomy group was induced more readily than in the sham‐operation group following UVB irradiation. These findings suggest that decreases in the estrogen levels after ovariectomy accelerate photoaging in terms of the morphology and physical properties of the skin surface and the three‐dimensional structure of elastic fibers.

Carbon dioxide laser treatment promotes repair of the three-dimensional network of elastic fibres in rat skin.

Background  We have previously reported that ultraviolet (UV) B irradiation induces a loss of linearity in the three‐dimensional structure of dermal elastic fibres, which results in the reduction of elastic properties of the skin and leads to wrinkle formation. We further reported that repair of wrinkles by all‐trans retinoic acid is accompanied by recovery of the linearity of elastic fibres. Carbon dioxide (CO2) lasers are widely used for treating wrinkles in cosmetic surgery.