Fermentable metabolite of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> controls collagen reduction in photoaging skin by improving TGF-β/Smad signaling suppression

Solar ultraviolet (UV) irradiation causes damages on human skin and premature skin aging (photoaging). UV-induced reduction of type I collagen in dermis is widely considered primarily induction of wrinkled appearance of photoaging skin. Type I procollagen synthesis is reduced under UV irradiation by blocking transforming growth factor-β (TGF-β)/Smad signaling; more specifically, it is down-regulation of TGF-β type II receptor (TβRII). Therefore, preventing UV-induced loss of TβRII results decreased type I collagen reduction in photoaging skin. Zymomonas mobilis is an alcohol fermentable, gram-negative facultative anaerobic bacterium whose effect on skin tissue is scarcely studied. We investigated the protective effects of fermentable metabolite of Z. mobilis (FM of Z. mobilis) against reduction of type I procollagen synthesis of UV-induced down-regulation of TβRII in human dermal fibroblasts FM of Z. mobilis was obtained from lyophilization of bacterium culture supernatant. The levels of TβRII and type I procollagen mRNA in human dermal fibroblasts were measured by quantitative real-time RT-PCR, and TβRII protein levels were assayed by western blotting. TβRII, type I procollagen, and type I collagen proteins in human dermal fibroblasts or hairless mouse skin were detected by immunostaining. FM of Z. mobilis inhibited down regulation of TβRII mRNA, and protein levels in UVB irradiated human dermal fibroblasts consequently recover reduced type I procollagen synthesis. These results indicate UVB irradiation inhibits type I procollagen synthesis by suppression of TGF-β/Smad signaling pathway, and FM of Z. mobilis has inhibitory effect on UVB-induced reduction of type I procollagen synthesis. While short period UVB irradiation decreased both TβRII and type I procollagen protein levels in hairless mouse skin, topical application of FM of Z. mobilis prevented this decrease. Wrinkle formation in hairless mouse skin surface was accelerated by continuous 5 month UVB irradiation along with a reduction of type I collagen in the dermis, but this change was prevented by topical application of FM of Z. mobilis. From this experimental data, it is suggested that FM of Z. mobilis is effective for suppression of wrinkle formation in photoaging skin by inhibition of type I procollagen synthesis reduction.     

Increase of collagen synthesis by obovatol through stimulation of the TGF-beta signaling and inhibition of matrix metalloproteinase in UVB-irradiated human fibroblast

BACKGROUND: Alterations of the extracellular matrix (ECM) is critical in the photo and age-damaged skin. Thus any compounds keep ECM can protected from photo and aged-damaged skin. ECM is predominantly composed of type I and type III collagens in the dermis. Transforming growth factor (TGF-beta)s play important roles in cellular biosynthesis of extracellular matrix. Activator protein 1 (AP-1) and Smad are significant factors that mediate TGF-beta. OBJECTIVE: We have investigated increasing effects of obovatol, a biphenolic compound isolated from leaves of Magnolia obovata on the collagen synthesis through stimulation of the TGF-beta signaling and inhibition of matrix metalloproteinase, thereby protect against from UV damages via maintain of collagen in the UVB irradiated human fibroblast cells. METHODS: The fibroblasts were pretreated with obovatol for 24h and then the cells were irradiated with UVB. UVB-exposed cells were further cultured for 24h. Type I procollagen, MMP-3, TGF-beta and Smad as well as phosphorylation of MAPK family expression were determined by Western blot. The activation of AP-1 was investigated using EMSA. The released type I procollagen and TGF-beta into cell culture medium were determined by Western blot after concentration of these proteins. RESULTS: The results showed that obovatol stimulated type I procollagen, TGF-beta, and Smad expression and inhibited matrix metalloproteinase-3 (MMP-3) in dose-dependent manner (1-5muM) in UVB-irradiated human fibroblast cells. Obovatol also inhibited UVB-induced activation of AP-1 and MAP kinases. CONCLUSION: These results suggest that obovatol increases collagen synthesis through stimulation of the TGF-beta signaling and inhibition of matrix metalloproteinase in UVB-irradiated human fibroblast, thus obovatol could be effective against photo-damaged skin.     

Extract of Punica granatum inhibits skin photoaging induced by UVB irradiation

Background Punica granatum (pomegranate) is kind of a fruit consumed fresh or in beverage. It has been widely used in traditional medicine in various parts of the world. In this study, we examined the efficacy of a Punica granatum (PG) extract in protecting skin against UVB‐induced damage using cultured human skin fibroblasts. Methods A Korean red PG sample was used, and its effects classified according to if the PG source originated from the rind, seed and fruit. The polyphenol content of PG, which is known to prevent other adverse cutaneous effects of UV irradiation, was measured by GC‐MS. The protective effects of PG on UVB‐induced skin photoaging were examined by determining the level of procollagen type I and MMP‐1 after UVB irradiation. Results Based on the GC‐MS quantitative analysis, catechin, quercetin, kaempferol, and equol were the predominant compounds detected in PG. In the changes of expression of procollagen type I and MMP‐1 in UV irradiated human skin fibroblasts treated PG, especially extract prepared from rind, the synthesis of collagen was increased and the expression of MMP‐1 was decreased. Conclusion The major polyphenols in PG, particularly catechin, play a significant role in its photoprotective effects on UVB‐induced skin damage.     

Inhibitors of dipeptidyl peptidase IV-like activity mediate antifibrotic effects in normal and keloid-derived skin fibroblasts

Suppression of collagen and matrix synthesis and inhibition of the fibrogenic cytokine transforming growth factor-beta(1) (TGF-beta(1)) is a major therapeutic goal in the treatment of fibrosis and keloids. Inhibitors of dipeptidyl peptidase IV (DP IV)-like activity affect cell growth and cytokine production and are currently under investigation for the treatment of metabolic, autoimmune and inflammatory diseases. We show here that the inhibitors of DP IV-like activity, Lys[Z(NO(2))]-thiazolidide and Lys[Z(NO(2))]-pyrrolidide, suppress proliferation in human skin fibroblasts and keloid-derived skin fibroblasts in vitro. They significantly decrease TGF-beta(1) expression and secretion of procollagen type I C-terminal peptide in supernatants of both cell types. Furthermore, they abrogate the TGF-beta(1)-induced stimulation of collagen synthesis, matrix deposition, and TGF-beta(1) and fibronectin expression. Both inhibitors lead to dephosphorylation of mitogen-activated protein kinases pp38 and pERK1/2, which are activated upon TGF-beta1 stimulation and have been implicated in fibrogenesis. In a mouse model of dermal fibrosis, induced by repetitive intracutaneous injections of TGF-beta(1), the profibrotic effect of TGF-beta(1) detected by dermal thickening, collagen I, and alpha-smooth muscle actin expression, is significantly suppressed in the presence of inhibitors. Inhibition of DP IV-like enzymatic activity may therefore represent a promising therapeutic approach for the treatment of fibrotic skin disorders and keloids.     

Modulation of skin collagen metabolism in aged and photoaged human skin in vivo.

To the best of our knowledge, no study has been conducted to date to directly compare the collagen metabolism of photoaged and naturally aged human skin. In this study, we compared collagen synthesis, matrix metalloproteinase-1 levels, and gelatinase activity of sun-exposed and sun-protected skin of both young and old subjects. Using northern blot analysis, immunohistochemical stain, and Western blot analysis, we demonstrated that the levels of procollagen type I mRNA and protein in photoaged and naturally aged human skin in vivo are significantly lower than those of young skin. Furthermore, we demonstrated, by northern blot analysis, that the procollagen alpha1(I) mRNA expression of photoaged skin is much greater than that of sun-protected skin in the same individual. In situ hybridization and immunohistochemical stain were used to show that the expression of type I procollagen mRNA and protein in the fibroblasts of photoaged skin is greater than for naturally aged skin. In addition, it was found, by Western blot analysis using protein extracted from the dermal tissues, that the level of procollagen type I protein in photoaged skin is lower than that of naturally aged skin. The level of matrix metalloproteinase-1 protein and the activity of matrix metalloproteinase-2 were higher in the dermis of photoaged skin than in naturally aged skin. Our results suggest that the natural aging process decreases collagen synthesis and increases the expression of matrix metalloproteinases, whereas photoaging results in an increase of collagen synthesis and greater matrix metalloproteinase expression in human skin in vivo. Thus, the balance between collagen synthesis and degradation leading to collagen deficiency is different in photoaged and naturally aged skin.     

Ultraviolet irradiation-induced inhibition of histone deacetylase 4 increases the expression of matrix metalloproteinase-1 but decreases that of type I procollagen via activating JNK in human dermal fibroblasts

BackgroundUltraviolet (UV) irradiation is the main contributing factor for skin aging. UV irradiation induces epigenetic changes in skin. It increases the activity of histone acetylases (HATs) but decreases that of histone deacetylases (HDACs).ObjectiveWe aimed to investigate alterations in all classes of HDACs and sirtuins (SIRTs) in response to UV irradiation, and determine the HDACs regulating the expressions of matrix metalloproteinase 1 (MMP-1) and type I procollagen.MethodsPrimary human dermal fibroblasts were UV irradiated. HDAC4 was knocked-down or overexpressed to investigate its effect on the expression of MMP-1 and type I procollagen. The mRNA and protein levels were analyzed by quantitative real-time polymerase chain reaction and western blotting.ResultsAmong 11 HDACs and 7 SIRTs, we found that the expression of HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC11, SIRT2, and SIRT3 were significantly and consistently reduced by UV at both mRNA and protein levels. Among these, the reduction of HDAC4 was responsible for the basal and UV-induced increase in the expression of MMP-1 and decrease in that of type I procollagen. Furthermore, the reduced HDAC4 could activate c-Jun N-terminal kinase (JNK), resulting in an increase in MMP-1 and decrease in type I procollagen.ConclusionsUV treatment decreases the expression of HDACs and SIRTs in dermal fibroblasts; in particular, the UV-induced reduction in the expression of HDAC4 might play an important role in regulating the expression of MMP-1 and type I procollagen.     

Inhibition of type I procollagen production in photodamage: correlation between presence of high molecular weight collagen fragments and reduced procollagen synthesis

Three-dimensional lattices of reconstituted, polymerized type I collagen were subjected to partial hydrolysis by organ culture fluid from human skin or by various matrix metalloproteinases, including matrix metalloproteinase-1 (interstitial collagenase), -2 (72 kDa gelatinase A), -8 (neutrophil collagenase), -9 (92 kDa gelatinase B), or -13 (collagenase 3). Following partial digestion, human dermal fibroblasts were incubated on the enzyme-treated or control lattices and examined for ability to contract the collagen lattice and synthesize type I procollagen. Collagen lattices partially degraded by organ culture fluid were contracted by fibroblasts under conditions in which control collagen lattices were not. On the partially degraded collagen, fibroblasts synthesized reduced amounts of type I procollagen (approximately 70% reduction). Purified matrix metalloproteinases with collagenolytic activity duplicated the effects of the human skin organ culture fluid, although matrix metalloproteinases 8 and 13 were less efficient than matrix metalloproteinase-1 (65% vs 40% and 18% reduction in type I procollagen production for matrix metalloproteinases 1, 8, and 13, respectively). Matrix metalloproteinases 2 and 9 were without effect on intact collagen; however, when collagen lattices were subjected to digestion by a combination of matrix metalloproteinases 1 and 9, fragments produced by matrix metalloproteinase-1 were further degraded by the gelatinase. Collagen contraction and inhibition of procollagen synthesis were both reduced. Matrix metalloproteinase-2 was less effective than matrix metalloproteinase-9 in clearing matrix metalloproteinase-1-generated fragments. Matrix metalloproteinase-2 was also less effective in preventing contraction and inhibiting the downregulation of type I procollagen synthesis. These observations suggest that in the presence of high molecular weight fragments of type I collagen, type I procollagen synthesis is inhibited. As these fragments are processed further, there is less inhibition of type I procollagen production.     

Antiwrinkle effect of adipose-derived stem cell: activation of dermal fibroblast by secretory factors

BACKGROUND: Adipose-derived stem cells (ADSC) have wound-healing and antioxidant effects on human skin via secretion of growth factors and activation of dermal fibroblasts. OBJECTIVE: Paracrine mechanism reducing ultraviolet-B (UVB)-induced wrinkles by ADSC is investigated in this study. METHODS AND RESULTS: Wrinkles were induced by an eight-week UVB irradiation, and were significantly improved by the subcutaneous injection of ADSC in hairless mice. In a replica analysis, parameters involving wrinkles were improved with mid-level and high doses of ADSC (1x10(4) and 1x10(5) cells). Dermal thickness and collagen contents in the dermis also were increased in the ADSC-injected groups. To characterize the paracrine mechanism involving the antiwrinkle effect of ADSC, a conditioned medium of ADSC (ADSC-CM) was directly incubated in human dermal fibroblasts (HDF). UVB irradiation reduced the proliferation of HDF, but this was reversed by the pretreatment of ADSC-CM in a dose-dependent manner. In a cell cycle analysis, ADSC-CM decreased the UVB-induced apoptotic cell death, which was demonstrated by the reduced sub-G1 phase of HDF. In addition, the ADSC-CM increased the protein expression of collagen type I and decreased the protein level of matrix metalloproteinase 1 in HDF, which may account for the increased collagen contents in the dermis. CONCLUSIONS: Collectively, these results indicate that the ADSC and its secretory factors are effective for UVB-induced wrinkles, and the antiwrinkle effect is mainly mediated by reducing UVB-induced apoptosis and stimulating collagen synthesis of HDF.     

UV-induced DNA damage initiates release of MMP-1 in human skin

Destruction of collagen is a hallmark of photoaging. The major enzyme responsible for collagen 1 digestion, matrix metalloproteinase-1 (MMP-1), is induced by exposure to sunlight. To study the molecular trigger for this induction, human skin was ultraviolet-B (UVB)-irradiated and treated with liposome-encapsulated DNA repair enzymes. The photolyase-mediated DNA repair of epidermal UV damage was associated with a reduction of MMP-1 mRNA and protein expression in both the epidermal and dermal compartments of the skin. The role of the epidermal cells in MMP-1 induction in the fibroblasts was examined when human epidermal keratinocytes were irradiated with UVB and their media were transferred to unirradiated human dermal fibroblasts. Transfer of media from irradiated keratinocytes to unirradiated fibroblasts enhanced MMP-1 mRNA and protein. Thus, UV damage to keratinocytes of the epidermis may participate in the destruction of collagen in the dermis by release of soluble mediators that signal fibroblasts to release MMP-1. The MMP-1 induction was reduced when the keratinocytes were treated with DNA repair enzymes T4 endonuclease V or UV endonuclease prior to transfer of the media to fibroblasts. This implies that UVB, which deposits most of its energy on the chromatin of the epidermal keratinocytes and to a lesser extent in the upper dermis, has a significant role in photoaging. DNA damage in the keratinocytes initiates one of the signals for MMP-1 release, and enhancing DNA repair can reduce MMP-1 expression in human skin cells and tissue.     

Exogenous nitric oxide enhances the synthesis of type I collagen and heat shock protein 47 by normal human dermal fibroblasts

BACKGROUND: It is well established that the alterations of dermal matrix contributes to skin aging characterized by wrinkles. On the other hand, physiological NO is useful to maintain skin homeostasis such as a vasodilatation. However, a role of NO on production of dermal matrix has been clarified. OBJECTIVE: In this study, we have attempted to analyze the role of NO on type I collagen synthesis of normal human dermal fibroblasts including expression of procollagen alphaI S(1) mRNA/protein and heat shock protein 47 (HSP47). METHODS: The effects of NO which was generated by two types of NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), on type I collagen and HSP47 and their related mRNA expression were examined with ELISA and RT-PCR. RESULTS: NO was significantly accelerated the production of type I collagen by fibroblasts corresponding with up-regulation of procollagen alphaI (1) mRNA. Furthermore, NO increased both levels of HSP47 protein and mRNA in fibroblasts in a dose-dependent manner. CONCLUSIONS: These results suggest that NO has dual effects on collagen synthesis by fibroblasts as follows; one is the direct stimulation of collagen synthesis due to the up-regulation of procollagen alphaI(1) mRNA, and the other is an indirect effect through the increase of HSP47 mRNA expression. This is the first report that exogenous NO stimulates HSP47 production by dermal fibroblasts.     

Adipose-derived stem cells as a new therapeutic modality for ageing skin

Stem cells are undifferentiated cells, which have the important properties of self-renewal and differentiation. Adipose-derived stem cells (ADSC) have relative advantages in accessibility and abundance compared to other kinds of stem cells. Regeneration therapy using ADSC has received attention in the treatment of various dermatologic diseases. In previous studies, ADSC were shown to have antioxidant, whitening and wound-healing effects in the skin through secretion of growth factors and by activating fibroblasts. In this study, we investigated whether ADSC could be used as an anti-ageing therapy, especially by dermal collagen synthesis and angiogenesis. Subcutaneous injection of ADSC significantly increased collagen synthesis in hairless mice, and dermal thickness, collagen density and fibroblast number also increased. In addition, procollagen type I protein and mRNA expression increased, which accounts for the increased dermal collagen density. Angiogenesis, which was visualized by CD31 and NG2 immunofluorescence stains, also increased in ADSC-treated skin. Our results suggest that ADSC therapy may be useful in ageing skin. Its effects are mainly mediated by stimulating collagen synthesis in dermal fibroblasts and increasing angiogenesis.     

Modulation of collagen metabolism by the topical application of dehydroepiandrosterone to human skin

Dehydroepiandrosterone (DHEA) and its sulfate conjugate (DHEA-S) are the most abundantly produced human adrenal steroids to be reduced with age. DHEA may be related to the process of skin aging through the regulation and degradation of extracelluar matrix protein. In this study, we demonstrate that DHEA can increase procollagen synthesis and inhibit collagen degradation by decreasing matrix metalloproteinases (MMP)-1 synthesis and increasing tisuue inhibitor of matrix metalloprotease (TIMP-1) production in cultured dermal fibroblasts. DHEA was found to inhibit ultraviolet (UV)-induced MMP-1 production and the UV-induced decrease of procollagen synthesis, probably due to the inhibition of UV-induced AP-1 activity. DHEA (5%) in ethanol:olive oil (1:2) was topically applied to buttock skin of volunteers 12 times over 4 weeks, and was found to significantly increase the expression of procollagen alpha1(I) mRNA and protein in both aged and young skin. On the other hand, topical DHEA significantly decreased the basal expression of MMP-1 mRNA and protein, but increased the expression of TIMP-1 protein in aged skin. We also found that DHEA induced the expressions of transforming growth factor-beta1 and connective tissue growth factor mRNA in cultured fibroblasts and aged skin, which may play a role in the DHEA-induced changes of procollagen and MMP-1 expression. Our results suggest the possibility of using DHEA as an anti-skin aging agent.     

瘢痕疙瘩成纤维细胞胶原合成的实验研究

目的 探讨瘢痕疙瘩中胶原过度聚集的原因.方法 从正常皮肤与活跃增生瘢痕疙瘩标本分别培养真皮成纤维细胞,采用³H-脯氨酸掺入法分别检测单层培养及胶原凝胶三维培养体系中成纤维细胞的胶原合成量.用斑点杂交法检测单层培养细胞的人前.α1(Ⅰ)型胶原mRNA水平.结果 瘢痕疙瘩成纤维细胞Ⅰ型胶原mRNA水平升高,其胶原合成量也显著高于正常真皮成纤维细胞(P<0.01).结论 在增生活跃瘢痕疙瘩中,成纤维细胞胶原合成功能处于活化状态,胶原合成增加是导致胶原过度积聚的重要原因.