Treatment Of Human Dermal Fibroblasts Research Articles

Synergistic interplay of UV radiation and urban particulate matter induces impairment of autophagy and alters cellular fate in senescence-prone human dermal fibroblasts.

Skin aging is a complex process influenced by intrinsic factors and environmental stressors, including ultraviolet (UV) radiation and air pollution, among others. In this study, we investigated the effects of UVA and UVB radiation, combined with urban particulate matter (UPM), on human dermal fibroblasts (HDF). We show here that treatment of HDF with a subcytotoxic dose of UVA/UVB results in a series of events leading to mitochondrial dysfunction, increased ROS levels, and DNA damage. These effects are known to trigger either cellular senescence or cell death, depending on the cells' ability to clear damage by activating autophagy. Whereas UPM treatment in isolation did not affect proliferation or survival of HDF, of note, simultaneous UPM treatment of UV-irradiated cells selectively inhibited autophagic flux, thereby changing cell fate of a fraction of the cell population from senescence to apoptotic cell death. Our findings highlight the synergistic effects of UV radiation and UPM on skin aging, emphasizing the need to consider these factors in assessing the impact of environmental stressors on human health and opening opportunities for developing comprehensive approaches to protect and preserve skin integrity in the face of growing environmental challenges.

Anti-senescence and anti-wrinkle activities of 3—bromo—4,5—dihydroxybenzaldehyde from Polysiphonia morrowii Harvey in human dermal fibroblasts

Objective: To investigate the anti-senescence effect of 3-bromo-4,5- dihydroxybenzaldehyde (BDB) from Polysiphonia morrowii Harvey in human dermal fibroblasts (HDF). Methods: HDF were subjected to treatment of BDB and then treated with hydrogen peroxide (H2O2) to induce premature senescence. Senescence-associated β-galactosidase (SA-β-gal) activity in HDF was determined using the SA-β-gal staining method. Intracellular reactive oxygen species (ROS) production was measured using the 2’,7’-dichlorodihydrofluorescein diacetate assay. Western blotting assay was performed to assess the level of antioxidant enzyme glutathione peroxidase 1 (GPX1). In addition, intracellular collagen and collagenase contents were analyzed using the respective ELISA kits. Elastase activity in HDF supernatants was measured from p-nitroaniline release and normalized using total protein content. Results: Treatment of HDF with H2O2 increased the activity of SAP-gal, but BDB pre-treatment resulted in the reduction of SA-β- gal activity. Moreover, BDB significantly reduced H2O2-induced intracellular ROS production. BDB also markedly increased the level of GPX1, which was inhibited by 400 µM of H2O2. Furthermore, in in vitro study, BDB significantly increased intracellular collagen content and decreased matrix metalloproteinase-1 and elastase activities in HDF. Conclusions: Our results demonstrate that BDB shows anti- senescence and anti-wrinkle activities in vitro.

Non pharmacological high-intensity ultrasound treatment of human dermal fibroblasts to accelerate wound healing

Inspired by the effectiveness of low-intensity ultrasound on tissue regeneration, we investigated the potential effect of short-term high-intensity ultrasound treatment for acceleration of wound healing in an in vitro wound model and dermal equivalent, both comprising human dermal fibroblasts. Short-term ultrasound of various amplitudes significantly increased the proliferation and migration of fibroblasts and subsequently increased the production of the extracellular matrix components fibronectin and collagen type I, both of which are important for wound healing and are secreted by fibroblasts. In addition, ultrasound treatment increased the contraction of a fibroblast-embedded three-dimensional collagen matrix, and the effect was synergistically increased in the presence of TGF-β. RNA-sequencing and bioinformatics analyses revealed changes in gene expression and p38 and ERK1/2 MAPK pathway activation in the ultrasound-stimulated fibroblasts. Our findings suggest that ultrasound as a mechanical stimulus can activate human dermal fibroblasts. Therefore, the activation of fibroblasts using ultrasound may improve the healing of various types of wounds and increase skin regeneration.

Antiphotoaging Effect of 3,5-Dicaffeoyl-epi-quinic Acid against UVA-Induced Skin Damage by Protecting Human Dermal Fibroblasts In Vitro.

Cutaneous aging is divided into intrinsic and exogenous aging correspondingly contributing to the complex biological phenomenon in skin. Intrinsic aging is also termed chronological aging, which is the accumulation of inevitable changes over time and is largely genetically determined. Superimposed on this intrinsic process, exogenous aging is associated with environmental exposure, mainly to ultraviolet (UV) radiation and more commonly termed as photoaging. UV-induced skin aging induces increased expression of matrix metalloproteinases (MMPs) which in turn causes the collagen degradation. Therefore, MMP inhibitors of natural origin are regarded as a primary approach to prevent or treat photoaging. This study investigated the effects of 3,5-dicaffeoyl-epi-quinic acid (DEQA) on photoaging and elucidated its molecular mechanisms in UVA-irradiated human dermal fibroblasts (HDFs). The results show that treatment with DEQA decreases MMP-1 production and increases type I collagen production in UVA-damaged HDFs. In addition, treatment of UVA-irradiated HDFs with DEQA downregulates MMP-1, MMP-3 and MMP-9 expression via blocking MAPK-cascade-regulated AP-1 transcriptional activity in UVA-irradiated HDFs. Furthermore, DEQA relieves the UVA-mediated suppression of type I procollagen and collagen expression through stimulating TGF-β/Smad signaling, leading to activation of the Smad 2/3 and Smad 4 nuclear translocation. These results suggest that DEQA could be a potential cosmetic agent for prevention and treatment of skin photoaging.

Prolonged treatment with Y-27632 promotes the senescence of primary human dermal fibroblasts by increasing the expression of IGFBP-5 and transforming them into a CAF-like phenotype.

The Rho-kinases (ROCK) inhibitor Y-27632 has been shown to promote the growth of epidermal cells, however, its potential effects on human dermal fibroblasts (HDFs) need to be clarified. Here we show that prolonged treatment of HDFs with Y-27632 decreased their growth by inducing senescence, which was associated with induction of the senescence markers p16 and p21, and downmodulation of the ERK pathways. The senescent HDFs induced by Y-27632 acquired a cancer-associated-fibroblast (CAF)-like phenotype to promote squamous cell carcinoma (SCC) cell growth in vitro. Expression of a newly identified target of Y-27632 by RNA-seq, insulin growth factor binding protein 5 (IGFBP-5), was dramatically increased after 24 h of treatment with Y-27632. Adding recombinant IGFBP-5 protein to the culture medium produced similar phenotypes of HDFs as did treatment with Y-27632, and knockdown of IGFBP-5 blocked the Y-27632-induced senescence. Furthermore, Y-27632 induced the expression of an IGFBP-5 upstream gene, GATA4, and knockdown of GATA4 also reduced the Y-27632-induced senescence. In summary, these results demonstrate for the first time that Y-27632 promotes cellular senescence in primary HDFs by inducing the expression of IGFBP-5 and that prolonged treatment with Y-27632 potentially transforms primary HDFs into CAF-like cells.

7‑MEGA™ 500 regulates the expression of COX‑2, MMP‑3 and type 1 procollagen in UVB‑irradiated human keratinocytes and dermal fibroblasts.

AlaskOmega® Omega 7 500, also known as Omega‑7 fatty acid or 7‑MEGA™, is a highly concentrated palmitoleic acid (C16:1). Little is known about how 7‑MEGA regulates skin inflammation and wrinkle formation in cultured skin cells. The present study aimed to investigate the effects of 7‑MEGA on the expression of cyclooxygenase‑2 (COX‑2), matrix metallopeptidase (MMP)‑1/3 and type 1 procollagen, which are markers of skin inflammation and wrinkle formation, in ultraviolet B (UVB)‑irradiated human dermal fibroblasts (HDFs) and keratinocytes (HaCaT). No toxicity was observed upon treatment of HDFs and HaCaT cells with 0.5‑2.5 µl/ml 7‑MEGA. The exposure of HaCaT cells to 10 mJ/cm2 UVB for 6 h resulted in increased protein and/or mRNA expression of COX‑2 and MMP‑3. Treatment of HaCaT cells with 2.5 µl/ml 7‑MEGA suppressed the UVB‑induced expression of COX‑2 and MMP‑3 in these cells. In addition, treatment with 2.5 µl/ml 7‑MEGA attenuated the UVB‑induced expression and phosphorylation levels of c‑Fos and c‑Jun, two components of the activator protein‑1 (AP‑1) transcription factor, in HaCaT cells. Exposure of HDFs to 60 mJ/cm2 UVB for 6 h significantly decreased the expression of type 1 procollagen protein, whereas treatment with 2.5 µl/ml 7‑MEGA partially reversed the effects of UVB on the expression of type 1 procollagen protein. These results demonstrated for the first time that 7‑MEGA regulated the expression of COX‑2, MMP‑3 and type 1 procollagen in UVB‑irradiated skin cells. The present study suggested that 7‑MEGA may serve as a novel agent against UVB‑induced skin inflammation and damage.

Artesunate inhibits myofibroblast formation via induction of apoptosis and antagonism of pro-fibrotic gene expression in human dermal fibroblasts.

The anti-malaria drug artesunate and other chemical analogs of artemisinin have demonstrated cytostatic and cytotoxic effects in bacterial and cancer cells. Artemisinin-derived compounds have also been demonstrated to attenuate fibrosis in preclinical animal models, but the mechanisms by which this inhibition occurs are not well-understood. We investigated the effects of artesunate on the emergence of the myofibroblast, which is causally implicated in pro-fibrotic pathologies. CRL-2097 human dermal fibroblasts were analyzed for protein and transcript expression after treatment with artesunate to analyze fibroblast activation. Proliferation and apoptosis were also evaluated following treatment with artesunate in this cell line. Treatment of human dermal fibroblasts with artesunate antagonized fibroblast activation and pro-fibrotic extracellular matrix (ECM) deposition, both at basal culture conditions and when cultured in the presence of exogenous transforming growth factor-β1 (TGF-β1), a major pro-fibrotic cytokine. Artesunate-treated fibroblasts also demonstrated decreased proliferation and increased apoptosis. Transcript analysis by quantitative real-time polymerase chain reaction demonstrated that artesunate downregulated expression of pro-fibrotic genes including canonical myofibroblast markers, ECM genes, and several TGF-β receptors and ligands, and upregulated expression of cell cycle inhibitors and matrix-metalloproteinases. Together, these data demonstrate that artesunate antagonizes fibroblast activation and decreases expression of pro-fibrotic genes, while also promoting myofibroblast apoptosis, suggesting that these mechanisms may be responsible in part for the anti-fibrotic effects of artesunate described previously.

Stereoisomer-specific ginsenoside 20(S)-Rg3 reverses replicative senescence of human diploid fibroblasts via Akt-mTOR-Sirtuin signaling

BackgroundThe replicative senescence of human dermal fibroblasts (HDFs) is accompanied by growth arrest. In our previous study, the treatment of senescent HDFs with Rg3(S) lowered the intrinsic reactive oxygen species (ROS) levels and reversed cellular senescence by inducing peroxiredoxin-3, an antioxidant enzyme. However, the signaling pathways involved in Rg3(S)-induced senescence reversal in HDFs and the relatedness of the stereoisomer Rg3(R) in corresponding signaling pathways are not known yet. MethodsWe performed senescence-associated β-galactosidase and cell cycle assays in Rg3(S)-treated senescent HDFs. The levels of ROS, adenosine triphosphate (ATP), and cyclic adenosine monophosphate (cAMP) as well as the mitochondrial DNA copy number, nicotinamide adenine dinucleotide (NAD)+/1,4-dihydronicotinamide adenine dinucleotide (NADH) ratio, and NAD-dependent sirtuins expression were measured and compared among young, old, and Rg3(S)-pretreated old HDFs. Major signaling pathways of phosphatidylinositol 3-kinase/Akt, 5' adenosine monophosphate-activated protein kinase (AMPK), and sirtuin 1/3, including cell cycle regulatory proteins, were examined by immunoblot analysis. ResultsGinsenoside Rg3(S) reversed the replicative senescence of HDFs by restoring the ATP level and NAD+/NADH ratio in downregulated senescent HDFs. Rg3(S) recovered directly the cellular levels of ROS and the NAD+/NADH ratio in young HDFs inactivated by rotenone. Rg3(S) mainly downregulated phosphatidylinositol 3-kinase/Akt through the inhibition of mTOR by cell cycle regulators like p53/p21 in senescent HDFs, whereas Rg3(R) did not alter the corresponding signaling pathways. Rg3(S)-activated sirtuin 3/PGC1α to stimulate mitochondrial biogenesis. ConclusionCellular molecular analysis suggests that Rg3(S) specifically reverses the replicative senescence of HDFs by modulating Akt-mTOR-sirtuin signaling to promote the biogenesis of mitochondria.

Periostin is induced by IL-4/IL-13 in dermal fibroblasts and promotes RhoA/ROCK pathway-mediated TGF-β1 secretion in abnormal scar formation

Excess scar formation can occur after skin injurふy and lead to abnormal scar formation, such as keloids and hypertrophic scars, which are characterised by substantial deposition of extracellular matrix in the dermis. Periostin, an extracellular matrix protein that plays a crucial role in skin development and maintaining homeostasis, is also involved in skin disorders such as systemic/limited scleroderma, wound closure, and abnormal scar formation. However, the mechanism of periostin involvement in abnormal scar formation is not yet fully understood. In this study, we investigated the mechanism by which periostin is involved in abnormal scar formation. Treatment of human dermal fibroblasts (HDFs) with IL-4 and IL-13, which are cytokines of Th2 type immune responses that are up-regulated in abnormal scars, dramatically elevated the levels of periostin mRNA and protein, and also promoted the secretion of periostin by HDFs. Transforming growth factor-β1 (TGF-β1) had the same effect on HDFs as IL-4 and IL-13. Stimulation of HDFs with periostin promoted RhoA/ROCK pathway-mediated TGF-β1 secretion from HDFs. Our results suggest that IL-4 and IL-13 induce periostin expression and secretion, and in turn, secreted periostin induces RhoA/ROCK pathway-mediated TGF-β1 secretion. Secreted TGF-β1 then induces further periostin production and secretion, thereby promoting abnormal scar formation.

In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers

A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea 12–17 and urea heterodimers 18–22, and to measure interference with type I and II topoisomerases. Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques (cell cycle distribution, detection of mitochondrial membrane potential dissipation, and analysis of metabolic activity/viability). The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14–17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells.

Visfatin Promotes Wound Healing through the Activation of ERK1/2 and JNK1/2 Pathway

Visfatin, a member of the adipokine family, plays an important role in many metabolic and stress responses. The mechanisms underlying the direct therapeutic effects of visfatin on wound healing have not been reported yet. In this study, we examined the effects of visfatin on wound healing in vitro and in vivo. Visfatin enhanced the proliferation and migration of human dermal fibroblasts (HDFs) and keratinocytes the expression of wound healing-related vascular endothelial growth factor (VEGF) in vitro and in vivo. Treatment of HDFs with visfatin induced activation of both extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinases 1 and 2 (JNK1/2) in a time-dependent manner. Inhibition of ERK1/2 and JNK1/2 led to a significant decrease in visfatin-induced proliferation and migration of HDFs. Importantly, blocking VEGF with its neutralizing antibodies suppressed the visfatin-induced proliferation and migration of HDFs and human keratinocytes, indicating that visfatin induces the proliferation and migration of HDFs and human keratinocytes via increased VEGF expression. Moreover, visfatin effectively improved wound repair in vivo, which was comparable to the wound healing activity of epidermal growth factor (EGF). Taken together, we demonstrate that visfatin promotes the proliferation and migration of HDFs and human keratinocytes by inducing VEGF expression and can be used as a potential novel therapeutic agent for wound healing.

Artesunate Antagonizes Myofibroblast Markers and Fibrosis‐Associated Extracellular Matrix Protein Expression in Human Dermal Fibroblasts

Artemisia annua, a plant species native to eastern Asia, is the source of a compound known as artemenisin that has been shown to have numerous medicinal effects. Artesunate, artemether and other chemical analogs of artemisinin, initially studied as antimalarial compounds, have also demonstrated cytostatic and cytotoxic effects in bacterial and cancer cells. Recently, artemisinin‐derivative compounds have also been demonstrated to attenuate fibrotic pathologies in various preclinical animal models, including those of hepatic and pulmonary fibrosis, but the mechanisms by which this inhibition of fibrosis occurs is not well‐understood. Given the ability of artemisinin derivatives, artesunate in particular, to antagonize various fibrotic pathologies, we decided to investigate the effects of artesunate on a process common to all fibrotic pathologies: the emergence of the highly secretory, contractile myofibroblast. We demonstrated by Western blot analysis that treatment of human dermal fibroblasts with artesunate results in a dose‐dependent decrease in expression of alpha smooth muscle actin (ɑ‐SMA), the de facto marker of fibroblast activation. Treatment of human dermal fibroblasts with artesunate also inhibited activation of fibroblasts mediated by the profibrotic cytokine TGF‐β1, which is largely responsible for differentiation of fibroblasts into myofibroblasts in vivo, and this antagonistic effect of artesunate towards fibroblast activation was consistent among multiple lines of primary human dermal fibroblasts. Further, immunofluorescent analysis for collagen I and collagen III demonstrated a marked reduction of both basal and TGF‐β1‐induced expression of extracellular matrix‐associated proteins. Understanding that fibrosis is the result of excessive differentiation of fibroblasts to myofibroblasts and accumulation of extracellular matrix proteins secreted by myofibroblasts, our data suggest that the antifibrotic mechanisms of artesunate in vivo may be mediated by their antagonistic effects towards myofibroblast phenotypes. We next plan to determine the direct mechanisms underlying the antagonism of artesunate towards fibroblast activation, paying particular attention to the hypotheses regarding the induction of myofibroblast apoptosis or cell cycle arrest, and the dependence of these phenotypes on the generation and activity of peroxide. With mechanistic data underpinning the effects of artesunate as an antagonist to profibrotic fibroblast activation, we hope that our work will enable progression towards the use of artesunate and other artemenisin‐derived compounds as therapies for fibrotic pathologies.Support or Funding InformationThis research was supported by a National Institutes of Health award to Tanja Dominko (grant # R01GM85456) and a National Science Foundation Integrative Graduate Education and Research Traineeship (grant number DGE 1144804) awarded to David Dolivo.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Fe3O4 Nanoparticles on Skin Tumor Cells and Dermal Fibroblasts.

Iron oxide (Fe3O4) nanoparticles have been used in many biomedical approaches. The toxicity of Fe3O4 nanoparticles on mammalian cells was published recently. Though, little is known about the viability of human cells after treatment with Fe3O4 nanoparticles. Herein, we examined the toxicity, production of reactive oxygen species, and invasive capacity after treatment of human dermal fibroblasts (HDF) and cells of the squamous tumor cell line (SCL-1) with Fe3O4 nanoparticles. These nanoparticles had an average size of 65 nm. Fe3O4 nanoparticles induced oxidative stress via generation of reactive oxygen species (ROS) and subsequent initiation of lipid peroxidation. Furthermore, the question was addressed of whether Fe3O4 nanoparticles affect myofibroblast formation, known to be involved in tumor invasion. Herein, Fe3O4 nanoparticles prevent the expression alpha-smooth muscle actin and therefore decrease the number of myofibroblastic cells. Moreover, our data show in vitro that concentrations of Fe3O4 nanoparticles, which are nontoxic for normal cells, partially reveal a ROS-triggered cytotoxic but also a pro-invasive effect on the fraction of squamous cancer cells surviving the treatment with Fe3O4 nanoparticles. The data herein show that the Fe3O4 nanoparticles appear not to be adequate for use in therapeutic approaches against cancer cells, in contrast to recently published data with cerium oxide nanoparticles.

Partial Somatic to Stem Cell Transformations Induced By Cell-Permeable Reprogramming Factors

The production of pluripotent stem cells (iPSCs) for therapeutic applications will require practical methods to achieve tight temporal and quantitative control of reprogramming factor (RF) expression, while avoiding the mutagenic potential of gene transfer. Toward this end, we have developed cell-permeable RF proteins (CP-RFs) incorporating newly developed macromolecule transduction domains (MTDs). Treatment of human dermal fibroblasts (HDFs) with combinations of cell-permeable OCT4, SOX2, KLF4, CMYC and either NANOG or LIN28 proteins induced the outgrowth of stem cell-like colonies (iSCs). iSC colonies generated with CP-RFs resembled embryonic stem cells with regard to morphology, biomarker expression, and extended capacity for self-renewal, but failed to expand as iPSC or ES cell lines. Partial reprogramming appears to be a common response to protein-based delivery of programming factors into somatic cells.

11β-Hydroxysteroid dehydrogenase blockade prevents age-induced skin structure and function defects

Glucocorticoid (GC) excess adversely affects skin integrity, inducing thinning and impaired wound healing. Aged skin, particularly that which has been photo-exposed, shares a similar phenotype. Previously, we demonstrated age-induced expression of the GC-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in cultured human dermal fibroblasts (HDFs). Here, we determined 11β-HSD1 levels in human skin biopsies from young and older volunteers and examined the aged 11β-HSD1 KO mouse skin phenotype. 11β-HSD1 activity was elevated in aged human and mouse skin and in PE compared with donor-matched photo-protected human biopsies. Age-induced dermal atrophy with deranged collagen structural organization was prevented in 11β-HSD1 KO mice, which also exhibited increased collagen density. We found that treatment of HDFs with physiological concentrations of cortisol inhibited rate-limiting steps in collagen biosynthesis and processing. Furthermore, topical 11β-HSD1 inhibitor treatment accelerated healing of full-thickness mouse dorsal wounds, with improved healing also observed in aged 11β-HSD1 KO mice. These findings suggest that elevated 11β-HSD1 activity in aging skin leads to increased local GC generation, which may account for adverse changes occurring in the elderly, and 11β-HSD1 inhibitors may be useful in the treatment of age-associated impairments in dermal integrity and wound healing.

(S)‐1‐α‐naphthylmethyl‐6,7‐dihydroxy‐1,2,3,4‐tetrahydroisoquinoline (CKD712), promotes wound closure by producing VEGF through HO‐1 induction in human dermal fibroblasts and mouse skin

Given the importance of VEGF and haem oxygenase (HO)-1 in wound healing, the present study tested the hypothesis that CKD712, a synthetic tetrahydroisoquinoline alkaloid, activated VEGF production through the induction of HO-1 in human dermal fibroblasts (HDFs) and in mouse skin to stimulate wound healing. Using HDFs, the effects of CKD712 on the production of VEGF and migration were evaluated. The mechanisms responsible were investigated using various signal inhibitors and small interfering RNA techniques. The ability of CKD712 to promote wound healing was also investigated in full-thickness skin-wounded mice. CKD712 treatment of HDFs increased VEGF production and accelerated migration, which was antagonized by anti-VEGF antibodies. Both an AMPK inhibitor (compound C) and a HO-1 activity inhibitor (SnPPIX) but not inhibitors of MAPKs, PI3K and PKC reduced the production of VEGF by CKD712. Interestingly, SnPPIX inhibited HO-1 expression but not p-AMPK, whereas compound C inhibited both p-AMPK and HO-1 induction by CKD712. Moreover, CKD712 decreased HO-1 expression without affecting the expression of p-AMPK by siHO-1 transfection, but it failed to induce HO-1 in siAMPKα1-transfected cells, suggesting that AMPK is involved in HO-1 induction by CKD712 in HDFs. Also, CKD712 shortened the time of wound closure in an SnPPIX-sensitive manner in a full-thickness skin-wounded mouse model. CKD712 accelerated cutaneous wound healing, at least in part, by the production of VEGF through HO-1 induction in HDFs and mouse skin.

Efficacy of a Novel Treatment Serum in the Improvement of Photodamaged Skin

SynopsisA novel treatment serum formulated to target multiple pathways in the anti-ageing cascade was tested both in vitro and in clinical settings. In vitro testing was performed to assess the ability to stimulate key proteins and genes fundamental to the anti-ageing cascade. The antioxidant potential of the formulation was studied in a UV-irradiation clinical study. A 12-week, open-label, single-centre study was conducted to determine whether this uniquely formulated topical treatment serum could improve visible signs of facial photodamage. Clinical evaluations showed statistically significant reductions in fine wrinkles and coarse wrinkles and improvements in skin texture, tone and radiance starting at week 4 with continued improvements at weeks 8 and 12. Subject self-assessments confirmed that the beneficial effects of the treatment serum were readily observed by the users. The treatment serum was well tolerated with no treatment-related adverse events reported during the 12-week study. Use of this novel treatment serum produced significant improvements in the visible signs of facial photodamage.RésuméUn nouveau sérum de traitement conçu pour cibler de multiples voies dans la cascade anti-âge a été testé à la fois in vitro et dans les conditions cliniques. Les tests in vitro ont été réalisés afin d'évaluer la capacité de stimuler les protéines et les gènes clés fondamentales de la cascade anti-vieillissement. Le potentiel antioxydant de la formulation a été étudié dans une étude clinique utilisant le rayonnement UV. Une étude de douze semaines, en mode ouvert, monocentrique a été menée afin de déterminer si ce sérum spécialement formulé pour le traitement topique peut améliorer les signes visibles du photo-vieillissement du visage. Des évaluations cliniques ont montré une réduction statistiquement significative des rides et des ridules secondaires et l'amélioration de la texture de la peau, du tonus et d'éclat à partir de la semaine 4 avec des améliorations continues aux semaines 8 et 12. Les autoévaluations par les sujets ont confirmé que les effets bénéfiques du sérum de traitement étaient facilement observés par les utilisateurs. Le sérum de traitement a été bien toléré avec aucun événement indésirable rapporté au cours de l'étude de 12 semaines. L'utilisation de ce nouveau sérum de traitement produit des améliorations significatives dans les signes visibles du photovieillissement du visage.

Protective Effects of Fermented Citrus Unshiu Peel Extract against Ultraviolet‐A‐induced Photoageing in Human Dermal Fibrobolasts

The aqueous extracts of Citrus unshiu peel containing flavonoid glycosides was used as co-substrate with Schizophyllum commune mycelia producing β-glucosidase and its biological activities were studied. β-glucosidase-produced S. commune mycelia converted the glycosides (narirutin and hesperidin) into aglycones (naringenin and hesperetin). The photoprotective potential of fermented C. unshiu peel extract with S. commune (S-CPE) was tested in human dermal fibroblasts (HDFs) exposed to UVA. It was revealed that S-CPE had an inhibitory effect on human interstitial collagenase (matrix metalloproteinase, MMP-1) expression in UVA-irradiated HDFs. The treatment of UVA-irradiated HDFs with S-CPE resulted in a dose-dependent decrease in the expression level of MMP-1 mRNA. The UVA irradiation raised the proportion of senescence-associated β-galactosidase (SA-β-gal) positive cells in comparison with the normal control group. The treatment of UVA-irradiated HDFs with S-CPE was shown to decrease the level of SA-β-gal (by approximately 45% at an S-CPE concentration 0.1%, w/v) compared with the UVA-irradiated HDFs. It was found that S-CPE containing hesperetin has notable collagen biosynthetic activity for fibroblasts, indicating that S-CPE can be promising cosmetic ingredients.

Fli1 Is a Negative Regulator of Estrogen Receptor α in Dermal Fibroblasts

Estrogen is an important regulator of dermal fibroblast functions, including extracellular matrix (ECM) synthesis. Estrogen mediates its effects through estrogen receptors (ERs), ERα and ERβ; however, regulation of ERs in dermal fibroblasts remains poorly understood. Friend leukemia integration factor 1 (Fli1), a member of the Ets transcription factor family, has been shown to play a pivotal role in regulation of the ECM genes in dermal fibroblasts. The aim of this study was to examine a possible interaction between Fli1 and estrogen pathways, focusing on ERα. We show that treatment of human dermal fibroblasts with transforming growth factor-β (TGF-β) increases ERα protein and mRNA levels. Similarly, ERα expression was increased in response to small interfering RNA (siRNA)-mediated depletion of Fli1, suggesting that Fli1 is a mediator of the TGF-β effects on ERα expression. Accordingly, we showed that Fli1 binds to the most proximal region of the ERα promoter, and dissociates from the promoter upon TGF-β treatment. An inverse correlation between Fli1 and ERα expression levels was confirmed in cultured skin fibroblasts obtained from Fli1(+/-) mice and in the skin of Fli1(+/-) mice in vivo. This study supports a role of Fli1 as a negative regulator of the ERα gene in dermal fibroblasts.

High-Density Lipoprotein (HDL) From Elderly and Reconstituted HDL Containing Glycated Apolipoproteins A-I Share Proatherosclerotic and Prosenescent Properties With Increased Cholesterol Influx

High-density lipoprotein (HDL) is a strong antioxidant, anti-inflammatory, and antisenescence molecule. However, in the current study, HDL from the elderly group (E-HDL) exhibited increased glycation with apolipoprotein (apo) A-I multimerization and decreased phospholipid content. Similarly, glycated apoA-I (gA-I) by fructosylation has a covalently multimerized band without a crosslinker and impaired phospholipid-binding ability. Treatment of human dermal fibroblasts and macrophages with E-HDL and gA-I caused more severe cellular senescence and foam cell formation, respectively; however, treatment with HDL from a young group (Y-HDL) and native apoA-I (nA-I) suppressed senescence and atherosclerosis. E-HDL(3) and reconstituted HDL (rHDL) containing gA-I showed enhanced cholesterol influx into macrophages compared with Y-HDL(3) and nA-I-rHDL. In conclusion, E-HDL and gA-I-rHDL share similar physiologic properties in macrophages and human dermal fibroblasts. E-HDL and gA-I-rHDL exacerbated cellular senescence and atherosclerosis with increased cellular cholesterol influx.