Senescence In Human Dermal Fibroblasts Research Articles

Derivation of Cell-Engineered Nanovesicles from Human Induced Pluripotent Stem Cells and Their Protective Effect on the Senescence of Dermal Fibroblasts.

Stem cells secrete numerous paracrine factors, such as cytokines, growth factors, and extracellular vesicles. As a kind of extracellular vesicle (EV), exosomes produced in the endosomal compartment of eukaryotic cells have recently emerged as a biomedical material for regenerative medicine, because they contain many valuable contents that are derived from the host cells, and can stably deliver those contents to other recipient cells. Although we have previously demonstrated the beneficial effects of human induced potent stem cell-derived exosomes (iPSC-Exo) on the aging of skin fibroblasts, low production yield has remained an obstacle for clinical applications. In this study, we generated cell-engineered nanovesicles (CENVs) by serial extrusion of human iPSCs through membrane filters with diminishing pore sizes, and explored whether the iPSC-CENV ameliorates physiological alterations of human dermal fibroblasts (HDFs) that occur by natural senescence. The iPSC-CENV exhibited similar characteristics to the iPSC-Exo, while the production yield was drastically increased compared to that of iPSC-derived EVs, including exosomes. The proliferation and migration of both young and senescent HDFs were stimulated by the treatment with iPSC-CENVs. In addition, it was revealed that the iPSC-CNEV restored senescence-related alterations of gene expression. Treatment with iPSC-CENVs significantly reduced the activity of senescence-associated-β-galactosidase (SA-β-Gal) in senescent HDFs, as well as suppressing the elevated expression of p53 and p21, key factors involved in cell cycle arrest, apoptosis, and cellular senescence signaling pathways. Taken together, these results suggest that iPSC-CENV could provide an excellent alternative to iPSC-exo, and be exploited as a resource for the treatment of signs of skin aging.

Substance P enhances cellular migration and inhibits senescence in human dermal fibroblasts under hyperglycemic conditions

Diabetes induces cellular dysfunction in dermal fibroblasts, such as impairment in migration, which is a major cause of chronic wound. Here, we demonstrated that the migration of human dermal fibroblasts was impaired under a high glucose culture condition. Substance P (SP) rescued the impaired migration of the fibroblasts. The activity of Rac1, Rho-associated kinase (ROCK), and Src was required for SP-mediated rescue of fibroblast migration. SP activated Rac1 and Src, whereas, NSC23766, a Rac1 inhibitor, and PP1 and PP2, Src inhibitors, inhibited SP-mediated enhancement of fibroblast migration. Y-27632, a ROCK inhibitor, inhibited the SP-mediated rescue of fibroblast migration. Senescence-associated β-galactosidase activity increased in human dermal fibroblasts cultured in a high glucose environment, but SP inhibited the β-galactosidase activity of the fibroblasts. These results suggest that SP promotes the migration of human dermal fibroblasts in diabetic-condition-mimicking cultures via the activity of Rac1, ROCK, and Src, and inhibits fibroblast senescence in hyperglycemic cultures.

Adipose-derived stem cell-conditioned medium protects fibroblasts at different senescent degrees from UVB irradiation damages.

Adipose-derived stem cells (ADSCs) and their derivatives have aroused intense interest in fields of dermatological and aesthetic medicine. As a major component detected in ADSCs secretome, platelet-derived growth factor AA (PDGF-AA) has been reported mediating extracellular matrix deposition and remodeling, thus might contribute to its anti-aging effect. On the basis of establishing an experimental model that simulate actual skin aging by exposing HDFs to both intrinsic and extrinsic aging factors, we pretreated human dermal fibroblasts (HDFs) with ADSC-conditioned medium (ADSC-CM) before being irradiated, aiming at exploring preventive effects of ADSCs secretome against aging damages. 48h after irradiation, we detected cellular proliferation; β-galactosidase stain; mRNA expressions of MMP-1, MMP-9, and TIMP-1; and protein expressions of collagen I, collagen III, and elastin. Moreover, we detected related protein expression of PI3K/Akt signal pathway, which can be activated by PDGF-AA and was newly found to promote extracellular matrix protein synthesis. Concentration of PDGF-AA in the prepared ADSC-CM decreased over time and maintained excellent bioactivity at low temperature until the 11th week. ADSC-CM pretreatment can slightly or significantly improve cellular proliferative activity and reduce cellular senescence in irradiated HDFs. Besides, ADSC-CM pretreatment increased collagen I, collagen III, elastin, and TIMP-1 expressions but decreased MMP-1 and MMP-9 expressions both in irradiated and nonirradiated HDFs. ADSC-CM pretreatment significantly increased pAkt protein expression, and ECM protein expression greatly decreased in case of LY294002 application. The results were similar in three generations of HDFs, yet varied with different degrees. Generally, ADSC-CM we prepared demonstrates a certain degree of positive role in preventing HDFs from intrinsic and extrinsic aging damages and that PDGF-AA may contribute to making it become effective with some other components in ADSC-CM.

Conditioned medium derived from human amniotic stem cells delays H2O2‑induced premature senescence in human dermal fibroblasts.

Stem cells derived from human amniotic membrane (hAM) are promising targets in regenerative medicine. A previous study focused on human amniotic stem cells in skin wound and scar-free healing. The present study aimed to investigate whether hydrogen peroxide (H2O2)-induced senescence of human dermal fibroblasts (hDFs) was influenced by the anti-aging effect of conditioned medium (CdM) derived from human amniotic stem cells. First, the biological function of two types of amniotic stem cells, namely human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs), on hDFs was compared. The results of cell proliferation and wound healing assays showed that CdM promoted cell proliferation and migration. In addition, CdM from hAECs and hAMSCs significantly promoted proliferation of senescent hDFs induced by H2O2. These results indicated that CdM protects cells from damage caused by H2O2. Treatment with CdM decreased senescence-associated β-galactosidase activity and improved the entry of proliferating cells into the S phase. Simultaneously, it was found that CdM increased the activity of superoxide dismutase and catalase and decreased malondialdehyde by reducing H2O2-induced intracellular reactive oxygen species production. It was found that CdM downregulated H2O2-stimulated 8-hydroxydeoxy-guanosine and γ-H2AX levels and decreased the expression of the senescence-associated proteins p21 and p16. In conclusion, the findings indicated that the paracrine effects derived from human amniotic stem cells aided delaying oxidative stress-induced premature senescence.

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.

499 Senescent fibroblasts accumulate lysophospholipids with immunomodulatory properties

With age, senescent cells accumulate in various tissues, which are causatively linked to a state of chronic inflammation and age-related functional decline. The senescence associated secretory phenotype (SASP), a cocktail of inflammatory cyto/chemokines and matrix proteases, majorly contributes to this phenomena. However, the knowledge on lipid mediators which we recently discovered as members of the SASP is very limited. We investigated whether the composition and amount of bioactive lipid mediators are altered in senescent fibroblasts. Using tandem mass spectrometry, we identified two lysophosphatidylcholines (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine; lyso-PCs) to be significantly elevated intracellularly in replicative-, and stress induced premature- senescence of human dermal fibroblasts and we were able to detect the same lipid species in cell supernatants. Telomerase transduced fibroblasts did not display elevated lyso-PCs even at more than seventy population doublings. When we exposed FB to lyso-PC, we found elevated expression and secretion of the chemokines CXCL1 and IL8. In monocyte lineage cells, however, lyso-PCs disturbed TLR2/6-CD36 signaling by the agonist FSL1 and uptake of labeled dextrane and therefore may interfere with the recognition of senescent cells. Thus, we propose that lyso-PCs contribute to the chronic pro-inflammatory environment not only directly, but also indirectly, by disturbing the clearance of senescent cells.

430 Targeting SASP exosomes to limit senescence-associated premature skin aging

There are increasing evidences that persistent senescent cells accumulating in tissues significantly participate to the gradual functional decline associated with aging. Senescent cells phenotype is characterized by an irreversible cell cycle arrest, an altered morphology and a modified metabolism and secretome. The SASP (senescence-associated secretory phenotype) encompass a large array of mediators (inflammatory cytokines, chemokines, proteases, reactive oxygen species…) that are believed to accelerate aging. Some non-coding RNAs (microRNAs or long noncoding RNAs) were recently identified as members of the SASP, and were shown to promote senescence induction and maintenance. Thus, we focused on exosomes, small vectorized extracellular vesicles that transport these noncoding RNAs (and other SASP constituents) and likely play a critical role in the long-distance propagation of SASP negative effects. Replicative senescence (RS) and stress-induced premature senescence (SIPS) were induced in human dermal fibroblasts (HDF) by repeated passage (P28) or exposure to hydrogen peroxide. Senescent phenotype and secretory profiles (nuclear markers, ß-galactosidase activity, cytokines, MMP-1…) were carefully characterized. In line with previous reports, we found that RS and SIPS fibroblasts produce much more exosomes than normal HDF. Several cargo microRNAs were identified by real-time quantitative PCR (miR-21, miR-23, miR-29 and miR-34). Exposure of normal HDF to isolated exosomes obtained from senescent HDF leads to metabolic changes, and chiefly collagen synthesis decline. This was not seen with exosomes from normal HDF. A vegetable extract enriched with wogonin, an identified inhibitor of SASP, could reduce exosomes production (other SASP constituents were also affected). The metabolic impact of senescent cells-derived exosomes was limited by the extract.

Substituting plant vegetative parts with callus cell extracts: Case study with Woodfordia fruticosa Kurz. – A potent ingredient in skin care formulations

Herbal pharmaceutical companies are dependent on plant parts which are used as raw materials in their products. The naturally available herbal raw material does not meet the demand of industries. This can lead to exploitation of nature and affect the biodiversity in various ways. In vitro plant micropropagation and conservation can be a successful alternative to minimize this environmental exploitation. Woodfordia fruticosa Kurz. (Lythraceae) is a plant endemic to India, known for its anti-aging benefits. Apical leaf explants were used to generate calli which was achieved in Murashige Skoog medium with 0.25 mg-l 2,4,5-T and 0.10 mg-l 6-BAP. Callus was sub cultured continuously for six months and retrieved every month for in vitro studies (WSC 1-WSC 6). Among them, WSC-3 (callus extract of third month) increased the collagen production by 60.67 ± 4.2% in human dermal fibroblasts (HDF). Collagen-I and elastin gene expression was increased in WSC-3 treated HDF. WSC-3 inhibited hydrogen peroxide (H2O2) induced cellular senescence in HDF. LC–MS analysis of WSC-3 upheld the presence of polyphenols like gallic acid, 1,2,3,4,6-pentagalloyl glucose, glucogallin, and other phytoconstituents. The results of the in vitro experiments were on par with the extracts obtained from flowers of W. fruticosa. IC50 values in HDF showed that callus cell extract is non-toxic (IC50: >1000 μg/ml) whereas, the flower extracts were moderately toxic (IC50 278.25 ± 1.85 μg/ml). Woodfordia fruticosa callus extract is a potential ingredient for skin care formulations, can be a strong substitute to conventional flower extract and warrants further clinical studies.

Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus.

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age-related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi-mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro-inflammatory cytokines and extracellular matrix-degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi-related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non-senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.

The Addition of Hyaluronic Acid into Platelet-Rich Fibrin Lysate in Restoration of Senescent Human Dermal Fibroblasts Activities

Senescent human dermal fibroblasts had reduced capacity in proliferation and collagen synthesis. It is due to unresponsiveness against transforming growth factor-β1 (TGF-β1) stimulation. Either platelet-rich fibrin (PRF)-lysate or hyaluronic acid (HA) can restore TGF-β1 signaling pathway.
 To determine whether HA addition to PRF lysate has a better activity than PRF-lysate alone in restoring senescent human dermal fibroblasts (HDFs) activities.
 HDF isolated from six different human skins was divided into normal HDFs and senescent HDFs which are induced by serum starvation. The senescent groups were then given 50% PRF-lysate and various levels of HA. Amelioration of TGF-β1 signaling was measured by cellular proliferation index and collagen deposition. 
 Addition of HA into PRF-lysate resulted in a significant increase in proliferation index and collagen deposition index than PRF-lysate alone. The best level of HA for this mixture ranged from 20.83 mM to 41.67 mM. HA in PRF lysate is an excellent candidate material for treating clinical signs related to senescent human dermal fibroblasts.
 
 Ethical permission: This experiment had gain approval from the local ethical committee, Ref: KE/FK/471/EC/2016 dated 17-05-2016.

The Clearance Effect of Tetrahedral DNA Nanostructures on Senescent Human Dermal Fibroblasts.

Senescent cells are characterized by their resistance to apoptosis, and upon their long-term survival senescent cells affect tissue function and eventually become deleterious to the organism. Thus, far, it has been gradually accepted that clearance of these senescent cells could reduce tissue dysfunction. This study aimed to investigate biological effects of tetrahedral DNA nanostructures (TDNs) on senescent cells. The results revealed a different biological effect of TDNs, and their clearance effect on senescent cells. TDNs can induce phenotypic changes in senescent cells, suppressing antiapoptotic BCL-2 family proteins and upregulating BAX, a BCL-2 family proapoptotic protein, to influence the expression levels and function of downstream proteins. Consequently, cytochrome C releasing promoted cleavage-mediated activation of pro-caspase-3 and its nuclear translocation from the cytoplasm to mediate apoptosis. The present results provide a foundation for further studies on the application of TDNs in studies on aging.

Involvement of ERK1/2 activation in the gene expression of senescence-associated secretory factors in human hepatic stellate cells.

Senescent hepatic stellate cells (senescent HSCs) are found in patients with liver cirrhosis and have been thought to be involved in the development of hepatocellular carcinoma (HCC) in mice via the senescence-associated secretory proteins. However, in humans, which secretory proteins are involved and what regulate their expression remain unclear. In the current study, we characterized senescence-associated β-galactosidase-positive senescent human HSCs (hHSCs) induced by repetitive passaging. They exhibited enhanced expression of 14 genes for secretory protein and persistent phosphorylation of ERK1/2 protein but not JNK or p38 MAPK proteins. Enhanced nuclear ERK1/2 phosphorylation was observed in senescent hHSCs. Treatment of the senescent hHSCs with ERK1/2 inhibitor, SCH772984, significantly decreased the levels of angiopoietin like 4 (ANGPTL4), C-C motif chemokine ligand 7 (CCL7), Interleukin-8 (IL-8), platelet factor 4 variant 1 (PF4V1), and TNF superfamily member 15 (TNFSF15) mRNA levels in a dose-dependent manner. The enhanced phosphorylation of ERK1/2 and expression of ANGPTL4, IL-8 and PF4V1 genes were observed in both of senescent human dermal fibroblasts and X-ray-induced senescent hHSCs. However, transient ERK1/2 activation induced by epidermal growth factor could not mimic the gene profile of the senescent hHSCs. These results revealed involvement of ERK1/2 signaling in the regulation of senescence-associated secretory factors, suggesting that simultaneous induction of ANGPTL4, IL-8, and PF4V1 genes is a marker of hHSC senescence. This study will contribute to understanding roles of senescent hHSCs in liver diseases.

Embryonic Stem Cell-Derived mmu-miR-291a-3p Inhibits Cellular Senescence in Human Dermal Fibroblasts Through the TGF-β Receptor 2 Pathway.

Senescent cells accumulate in various tissues over time and contribute to tissue dysfunction and aging-associated phenotypes. Accumulating evidence suggests that cellular senescence can be inhibited through pharmacological intervention, as well as through treatment with soluble factors derived from embryonic stem cells (ESCs). In an attempt to investigate the anti-senescence factors secreted by ESCs, we analyzed mouse ESC-derived extracellular microRNAs in conditioned medium via microRNA array analysis. We selected mmu-miR-291a-3p as a putative anti-senescence factor via bioinformatics analysis. We validated its inhibitory effects on replicative, Adriamycin-induced, and ionizing radiation-induced senescence in human dermal fibroblasts. Treatment of senescent cells with mmu-miR-291a-3p decreased senescence-associated β-galactosidase activity, enhanced proliferative potential, and reduced mRNA and protein expression of TGF-β receptor 2, p53, and p21. mmu-miR-291a-3p in conditioned medium was enclosed in ESC-derived exosomes and exosomes purified from ESC conditioned medium inhibited cellular senescence. The inhibitory effects of mmu-miR-291a-3p were mediated through the TGF-β receptor 2 signaling pathway. Hsa-miR-371a-3p and hsa-miR-520e, the human homologs of mmu-miR-291a-3p, showed similar anti-senescence activity. Furthermore, mmu-miR-291a-3p accelerated the excisional skin wound healing process in aged mice. Our results indicate that the ESC-derived mmu-miR-291a-3p is a novel candidate agent that can be utilized for cell-free therapeutic intervention against aging and aging-related diseases.

TLR4-mediated activation of the ERK pathway following UVA irradiation contributes to increased cytokine and MMP expression in senescent human dermal fibroblasts.

Exposure to ultraviolet (UV) radiation is a major contributing factor to premature aging (photoaging) and skin cancer. In vitro models of cellular senescence have proven to be very useful for the study of slow and progressive accumulation of damage resulting in the growth arrest of aging skin cells. In this study, we compared UVA-induced cellular responses in non-senescent (NS) vs. senescent (S) human dermal fibroblasts (HDFs). HDFs were irradiated with a single dose of UVA (7.5 J/cm2) and QuantSeq 3' mRNA sequencing was performed to assess differential gene expression. Both NS and S HDFs expressed similar numbers of differentially expressed genes, although distinct sets of genes were differentially expressed between the two groups. Higher expression of matrix metalloproteinases (MMPs) and Toll-like receptor (TLR) pathway genes, such as TLR4, MyD88, and CXCL-8, was detected in S HDFs as compared with NS HDFs, and UVA exposure led to a downregulation of collagen genes, such as COL8A2 and COL5A3. Consistent with gene expression profiling, enhanced IL-6 and IL-8 secretion was observed in S HDFs compared with NS HDFs, in response to UVA. Furthermore, we show that TLR4-mediated ERK pathway is responsible for the UVA-mediated mitochondrial dysfunction as well as increased secretion of MMP-1 and IL-8 in S HDFs. Taken together, our results demonstrate the UVA-induced common and distinct molecular patterns of cellular responses between NS and S HDFs and suggest TLR4/ERK pathways as candidate targets to reduce senescent phenotypes.

Proteomic analyses reveal that ginsenoside Rg3(S) partially reverses cellular senescence in human dermal fibroblasts by inducing peroxiredoxin

BackgroundThe cellular senescence of primary cultured cells is an irreversible process characterized by growth arrest. Restoration of senescence by ginsenosides has not been explored so far. Rg3(S) treatment markedly decreased senescence-associated β-galactosidase activity and intracellular reactive oxygen species levels in senescent human dermal fibroblasts (HDFs). However, the underlying mechanism of this effect of Rg3(S) on the senescent HDFs remains unknown. MethodsWe performed a label-free quantitative proteomics to identify the altered proteins in Rg3(S)-treated senescent HDFs. Upregulated proteins induced by Rg3(S) were validated by real-time polymerase chain reaction and immunoblot analyses. ResultsFinally, 157 human proteins were identified, and variable peroxiredoxin (PRDX) isotypes were highly implicated by network analyses. Among them, the mitochondrial PRDX3 was transcriptionally and translationally increased in response to Rg3(S) treatment in senescent HDFs in a time-dependent manner. ConclusionOur proteomic approach provides insights into the partial reversing effect of Rg3 on senescent HDFs through induction of antioxidant enzymes, particularly PRDX3.

Anti-inflammatory activities of Ophiopogonis Radix on hydrogen peroxide-induced cellular senescence of normal human dermal fibroblasts.

Ophiopogonis Radix (Ophiopogon root), which nourishes the yin, has been used in clinical practice to promote fluid secretion and to moisturize the lungs and skin in traditional Chinese and Japanese (Kampo) medicine. To evaluate this traditional medicinal effect, we investigated the anti-chronic inflammatory effect of Radix Ophiopogonis on senescent cells. Conversely, although several phenotypes of Ophiopogon japonicus Ker-Gawler (Liliaceae) are prevalent in Japan and China, we used these Ophiopogon roots by considering them as one crude drug, Ophiopogonis Radix. In this study, it was revealed that there are two chemotypes (Types A and B) in the root of the original plant, O. japonicus. Methylophiopogonanone A (compound 1) and methylophiopogonanone B (compound 2) were isolated as index compounds from Type A and compound 1 and ophiopogonanone A (compound 3) from Type B. In addition, ophiopogonin B (compound 4) was isolated as the main steroidal saponin from both Type A and B. The results indicated that two different methanol extracts (from Types A and B) and the main constituents of O. japonicus (compound 1-4), significantly downregulated the expression of interleukin (IL)-6 and IL-8, which were enhanced by senescent normal human dermal fibroblasts. Moreover, the two different methanol extracts and compounds 1-4 decreased IL-6 production in a strong and concentration-dependent manner by the ELISA method.

Bovine vitreous gel can reactivate replicative senescence of human dermal fibroblast

The most influential factor in the poor healing of chronic ulcers is replicative senescence of fibroblasts that are unresponsive to TGF-β1 stimulation. Cellular replicative senescence can be induced by cultivating normal human dermal fibroblasts (HDFs) in a serum-starved medium. In addition, increasing microenviroment mechanical forces by hyaluronic acid can ameliorate the TGF-β1 signaling of these senescent cells. One of natural resources of hyaluronic acid is bovine vitreous gel. In order to evaluate the effect of bovine-vitreous gel on replicative senescence of fibroblasts, we used various levels of bovine vitreous gel diluted in Dulbecco’s modified Eagle’s medium to stimulate cellular activities of serum-starved HDFs. Those cellular activities were compared to the control media, standardized hyaluronic acid, and to normal HDFs. Our results show that replicative senescence of HDFs treated with 50% bovine vitreous gel exhibited a significantly higher proliferation index, migration rate, and collagen deposition than those cultured in control media, and they displayed an equal level of cellular activity with the HDFs exposed only to standardized hyaluronic acid. We concluded that bovine vitreous gel can be used to stimulate replicative senescence of HDFs and therefore a potential candidate material to stimulate healing of chronic ulcers.

Exosomes Derived from Human Induced Pluripotent Stem Cells Ameliorate the Aging of Skin Fibroblasts.

Stem cells and their paracrine factors have emerged as a resource for regenerative medicine. Many studies have shown the beneficial effects of paracrine factors secreted from adult stem cells, such as exosomes, on skin aging. However, to date, few reports have demonstrated the use of exosomes derived from human pluripotent stem cells for the treatment of skin aging. In this study, we collected exosomes from the conditioned medium of human induced pluripotent stem cells (iPSCs) and investigated the effect on aged human dermal fibroblasts (HDFs). Cell proliferation and viability were determined by an MTT assay and cell migration capacity was shown by a scratch wound assay and a transwell migration assay. To induce photoaging and natural senescence, HDFs were irradiated by UVB (315 nm) and subcultured for over 30 passages, respectively. The expression level of certain mRNAs was evaluated by quantitative real-time PCR (qPCR). Senescence-associated-β-galactosidase (SA-β-Gal) activity was assessed as a marker of natural senescence. As a result, we found that exosomes derived from human iPSCs (iPSCs-Exo) stimulated the proliferation and migration of HDFs under normal conditions. Pretreatment with iPSCs-Exo inhibited the damages of HDFs and overexpression of matrix-degrading enzymes (MMP-1/3) caused by UVB irradiation. The iPSCs-Exo also increased the expression level of collagen type I in the photo-aged HDFs. In addition, we demonstrated that iPSCs-Exo significantly reduced the expression level of SA-β-Gal and MMP-1/3 and restored the collagen type I expression in senescent HDFs. Taken together, it is anticipated that these results suggest a therapeutic potential of iPSCs-Exo for the treatment of skin aging.

Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype.

Loss of functionality during aging of cells and organisms is caused and accompanied by altered cell-to-cell communication and signalling. One factor thereby is the chronic accumulation of senescent cells and the concomitant senescence-associated secretory phenotype (SASP) that contributes to microenvironment remodelling and a pro-inflammatory status. While protein based SASP factors have been well characterized, little is known about small extracellular vesicles (sEVs) and their miRNA cargo. Therefore, we analysed secretion of sEVs from senescent human dermal fibroblasts and catalogued the therein contained miRNAs. We observed a four-fold increase of sEVs, with a concomitant increase of >80% of all cargo miRNAs. The most abundantly secreted miRNAs were predicted to collectively target mRNAs of pro-apoptotic proteins, and indeed, senescent cell derived sEVs exerted anti-apoptotic activity. In addition, we identified senescence-specific differences in miRNA composition of sEVs, with an increase of miR-23a-5p and miR-137 and a decrease of miR-625-3p, miR-766-3p, miR-199b-5p, miR-381-3p, miR-17-3p. By correlating intracellular and sEV-miRNAs, we identified miRNAs selectively retained in senescent cells (miR-21-3p and miR-17-3p) or packaged specifically into senescent cell derived sEVs (miR-15b-5p and miR-30a-3p). Therefore, we suggest sEVs and their miRNA cargo to be novel, members of the SASP that are selectively secreted or retained in cellular senescence.

Fluorofenidone inhibits UV-A induced senescence in human dermal fibroblasts via the mammalian target of rapamycin-dependent SIRT1 pathway.

The aim of this study was to investigate the protective effect of fluorofenidone (5‐methyl‐1‐[3‐fluorophenyl]‐2‐[1H]‐pyridone, AKF‐PD) on ultraviolet (UV)‐A‐induced senescence in human dermal fibroblasts (HDF) and examine the mechanisms involved. HDF were treated with AKF‐PD. Senescence‐associated (SA)‐β‐galactosidase level, cell viability and expression of p16 were evaluated. In addition, UV‐A‐irradiated HDF were treated with AKF‐PD, rapamycin and MHY1485; SA‐β‐galactosidase staining, 3‐(4 5‐dimethylthiazol‐2‐yl)‐2 5‐diphenyltetrazolium bromide assay and western blot for SIRT1 were performed; and phosphorylated mammalian target of rapamycin (p‐mTOR) expression and reactive oxygen species (ROS) levels were measured. Intracellular ROS was detected by the 2′,7′‐dichlorofluroescein diacetate probe. Our results showed that AKF‐PD substantially attenuated the changes of p16 expression, SA‐β‐galactosidase staining and cellular proliferation induced by UV‐A irradiation in HDF. AKF‐PD rescued the increased mTOR phosphorylation and reduced SIRT1 expression induced by UV‐A irradiation in HDF. AKF‐PD and rapamycin together had a synergistic effect on p‐mTOR reduction and SIRT1 increase. mTOR activator MHY1485 partly blocked the above effects. Moreover, intracellular ROS level induced by UV‐A irradiation could partly decrease by AKF‐PD, and MHY1485 could reduce this effect. Our results indicated that AKF‐PD could alleviate HDF senescence induced by UV‐A‐irradiation by inhibiting the p‐mTOR and increasing SIRT1. Moreover, AKF‐PD may be a potential treatment material for skin.