Senescent Human Fibroblasts Research Articles

Remodeling of cytoskeleton, chromatin and gene expression during mechanical rejuvenation of aged human dermal fibroblasts.

Aging is associated with a progressive decline in cellular function. To reset the aged cellular phenotype, various reprogramming approaches, including mechanical routes, have been explored. However, the epigenetic mechanisms underlying cellular rejuvenation are poorly understood. Here, we studied the cytoskeletal, genome-wide chromatin and transcriptional changes in young, aged and mechanically rejuvenated fibroblasts using immunofluorescence, RNA-seq and Hi-C experiments. The mechanically rejuvenated aged fibroblasts, that had partially reset their transcription to a younger cell state, showed a local reorganization of the inter-chromosomal contacts and lamina-associated domains. Interestingly, the observed chromatin reorganization correlated with the transcriptional changes. Immunofluorescence experiments in the rejuvenated state confirmed increased acto-myosin contractility like younger fibroblasts. In addition, the rejuvenated contractile properties were maintained over multiple cell passages. Overall, our results give an overview of how changes in the cytoskeleton, chromatin, and gene activity are connected to aging and rejuvenation.

Design of a Magnetic Nanoplatform Based on CD26 Targeting and HSP90 Inhibition for Apoptosis and Ferroptosis-Mediated Elimination of Senescent Cells.

The accumulation of senescent cells, a hallmark of aging and age-related diseases, is also considered as a side effect of anticancer therapies, promoting drug resistance and leading to treatment failure. The use of senolytics, selective inducers of cell death in senescent cells, is a promising pharmacological antiaging and anticancer approach. However, more studies are needed to overcome the limitations of first-generation senolytics by the design of targeted senolytics and nanosenolytics and the validation of their usefulness in biological systems. In the present study, we have designed a nanoplatform composed of iron oxide nanoparticles functionalized with an antibody against a cell surface marker of senescent cells (CD26), and loaded with the senolytic drug HSP90 inhibitor 17-DMAG (MNP@CD26@17D). We have documented its action against oxidative stress-induced senescent human fibroblasts, WI-38 and BJ cells, and anticancer drug-induced senescent cutaneous squamous cell carcinoma A431 cells, demonstrating for the first time that CD26 is a valid marker of senescence in cancer cells. A dual response to MNP@CD26@17D stimulation in senescent cells was revealed, namely, apoptosis-based early response (2 h treatment) and ferroptosis-based late response (24 h treatment). MNP@CD26@17D-mediated ferroptosis might be executed by ferritinophagy as judged by elevated levels of the ferritinophagy marker NCOA4 and a decreased pool of ferritin. As 24 h treatment with MNP@CD26@17D did not induce hemolysis in human erythrocytes in vitro, this newly designed nanoplatform could be considered as an optimal multifunctional tool to target and eliminate senescent cells of skin origin, overcoming their apoptosis resistance.

Fifty-hertz magnetic fields induce DNA damage through activating mPTP associated mitochondrial permeability transition in senescent human fetal lung fibroblasts

With the rapid development and using of electromagnetic technology, artificial electromagnetic fields (EMFs) have become an emerging environmental factor in our daily life. Extremely-low-frequency (ELF) magnetic fields (MFs), generally generated by power lines and various electric equipment, is one of the most common EMFs in the environment which were concerned for the potential impact on human health. Base on limited evidence, ELF-MFs have been classified as possible carcinogen to human by International Agency for Research on Cancer (IARC), but the mechanisms have not been fully elucidated. Senescent cells are a group of special cells, characterized by cell cycle arrest, senescence-associated secretory phenotype (SASP), accumulation of macromolecular damage, and metabolic disturbance, play important role in fetal development, tissue aging, and even carcinogenesis. Thus, EMFs may promote carcinogenesis by affecting senescent cells, however, there are few studies. In this study, we found that exposure to 50 Hz MFs at 1.0 mT for 24 h could induce significant DNA damage in senescent but not non-senescent human fetal lung fibroblast suggested that senescent cells are more sensitive to 50 Hz MFs on DNA damage, and further results revealed that reactive oxygen species (ROS) generation mediated by mitochondrial permeability transition pore (mPTP) activation play critical role in this process. Our results indicated that cellular senescence can lead to cell sensitivity to the DNA damage effect of 50 Hz MFs, however, whether this play important role in mediating the carcinogenesis of EMFs await further study.

Quantitative determination of the spatial distribution of components in single cells with CellDetail

The distribution of biomolecules within cells changes upon aging and diseases. To quantitatively determine the spatial distribution of components inside cells, we built the user-friendly open-source 3D-cell-image analysis platform Cell Detection and Analysis of Intensity Lounge (CellDetail). The algorithm within CellDetail is based on the concept of the dipole moment. CellDetail provides quantitative values for the distribution of the polarity proteins Cdc42 and Tubulin in young and aged hematopoietic stem cells (HSCs). Septin proteins form networks within cells that are critical for cell compartmentalization. We uncover a reduced level of organization of the Septin network within aged HSCs and within senescent human fibroblasts. Changes in the Septin network structure might therefore be a common feature of aging. The level of organization of the network of Septin proteins in aged HSCs can be restored to a youthful level by pharmacological attenuation of the activity of the small RhoGTPase Cdc42.

Sulfated galactans ameliorate the cellular senescence in dermal fibroblast cells

Replicative senescence of dermal fibroblast is the primary mechanism of intrinsic skin aging. Recent research has focused on the different properties of sulfated galactans (SG) derived from the red seaweed Gracilaria fisheri. Therefore, we aimed to investigate the potential impact of SG on anti-aging skin. This study used a validated replicative senescent human dermal fibroblast (HDF) model. We observed that replicative senescent HDF cells exhibited remarkable changes in cell morphology, decreased cell proliferation, retarded migrating activity, and reduced extracellular matrix (ECM) production. Additionally, the replicative senescent HDF cells exhibited increased expression of cell cycle inhibitors p16 and p21. Our results demonstrated that SG is safe and effective in reversing these morphological and molecular alterations in senescent HDF cells. SG mediated enhanced ECM synthesis, including collagen, elastin, and hyaluronic acid (HA), and promoted cell migration by upregulating TGF-β1 and TLP gene expression. SG also reduced the expression of ECM breakdown enzymes MMP-1, MMP-2, MMP-3, and MMP-9 while increasing MMP inhibitors TIMP-1 and TIMP-2. These results indicate that SG effectively restores fibroblast function, contributing to decelerating the aging processes, supporting its potential as a therapeutic agent for skin rejuvenation and anti-aging treatments.

SF3B4 Regulates Cellular Senescence and Suppresses Therapy-induced Senescence of Cancer Cells.

Cellular senescence is a state in which cells permanently exit the cell cycle, preventing tumor growth, but it can also contribute to aging and chronic inflammation. Senescence induced by cancer therapies, known as therapy-induced senescence (TIS), halts cancer cell proliferation and prevents metastasis. TIS has been investigated as an important therapeutic approach that could minimize cytotoxicity effects. This study aimed to elucidate the role of splicing factor 3B subunit 4 (SF3B4) in cellular senescence and TIS in cancer cells. β-galactosidase staining was used to examine senescence induction. SF3B4 and p21 expression were determined by RT-qPCR and western blot. Cell proliferation and cell death were evaluated. SF3B4 expression decreases in replicative senescent human fibroblasts and its knockdown induces senescence via a p21-dependent pathway. In A549 non-small cell lung cancer (NSCLC) cells, SF3B4 knockdown also increased senescence markers. Notably, SF3B4 overexpression mitigated doxorubicin-induced senescence in A549 cells. SF3B4 regulates senescence, and this study highlights its potential as a therapeutic target for developing better cancer treatment strategies by leveraging TIS to suppress tumor growth and enhance treatment efficacy.

Divergent regulation of long non-coding RNAs H19 and PURPL affects cell senescence in human dermal fibroblasts.

Cellular senescence is a permanent cell growth arrest that occurs in response to various intrinsic and extrinsic stimuli and is associated with cellular and molecular changes. Long non-coding RNAs (lncRNAs) are key regulators of cellular senescence by affecting the expression of many important genes involved in senescence-associated pathways and processes. Here, we evaluated a panel of lncRNAs associated with senescence for their differential expression between young and senescent human dermal fibroblasts (NHDFs) and studied the effect of a known senomorphic compound, resveratrol, on the expression of lncRNAs in senescent NHDFs. As markers of senescence, we evaluated cell growth, senescence-associated (SA)-β-Gal staining, and the expression of p21, Lamin B1 and γH2AX. We found that H19 and PURPL were the most altered lncRNAs in replicative, in doxorubicin (DOXO) and ionising radiation (IR)-induced senescence models. We then investigated the function of H19 and PURPL in cell senescence by siRNA-mediated silencing in young and senescent fibroblasts, respectively. Our results showed that H19 knockdown reduced cell viability and induced cell senescence and autophagy of NHDFs through the regulation of the PI3K/AKT/mTOR pathway; conversely, PURPL silencing reversed senescence by reducing (SA)-β-Gal staining, recovering cell proliferation with an increase of S-phase cells, and reducing the p53-dependent DNA damage response. Overall, our data highlighted the role of H19 and PURPL in the senescent phenotype and suggested that these lncRNAs may have important implications in senescence-related diseases.

A Two-Photon-Active Zr-Based Metal-Organic Framework-Based Orthogonal Nanoprobe for Recognition of Cellular Senescence.

A luminescent nanoprobe capable of orthogonal sensing of two independent events is highly significant for unbiased disease-related detection such as the detection of senescent cells. Moreover, it is invaluable that the nanoprobe possesses a two-photon excitable characteristic that is highly suitable for imaging living cells and tissues. Herein, we present a two-photon-excitable multiluminescent orthogonal-sensing nanoprobe (OS nanoprobe) capable of detecting both pH elevation and β-galactosidase (β-gal) overexpression in senescent cells. In the design, Zr-based dual-emissive metal-organic frameworks prepared from two mixed amino linkers, referred to as NH2-MU, were used as the component for the ratiometric sensing of pH; additionally, fluorogenic resorufin-β-d-galactopyranoside, linked to the NH2-MU framework, enables β-gal detection. In the OS nanoprobe, the signals for pH and β-gal sensing remain independent while maintaining high colocalization. The two-photon excitable organic linkers of NH2-MU impart the OS nanoprobe with a bioimaging capability, allowing for the differentiation of senescent human foreskin fibroblast (HFF) cells from younger HFF cells or LacZ positive cells with the 800 nm laser excitation. This study marks the first instance of achieving the multiplexed orthogonal fluorescent sensing of cellular senescence using a two-photon excitation strategy, suggesting the potential of using versatile metal-organic framework (MOFs)-based fluorophores to realize the orthogonal multiplexing of disease-related biomarkers through multiphoton excitation.

Systemic comparison of molecular characteristics in different skin fibroblast senescent models.

Senescent human skin primary fibroblast (FB) models have been established for studying aging-related, proliferative, and inflammatory skin diseases. The aim of this study was to compare the transcriptome characteristics of human primary dermal FBs from children and the elderly with four senescence models. Human skin primary FBs were obtained from healthy children (FB-C) and elderly donors (FB-E). Senescence models were generated by ultraviolet B irradiation (FB-UVB), D-galactose stimulation (FB-D-gal), atazanavir treatment (FB-ATV), and replication exhaustion induction (FB-P30). Flow cytometry, immunofluorescence staining, real-time quantitative polymerase chain reaction, co-culturing with immune cells, and bulk RNA sequencing were used for systematic comparisons of the models. In comparison with FB-C, FB-E showed elevated expression of senescence-related genes related to the skin barrier and extracellular matrix, proinflammatory factors, chemokines, oxidative stress, and complement factors. In comparison with FB-E, FB-UVB and FB-ATV showed higher levels of senescence and expression of the genes related to the senescence-associated secretory phenotype (SASP), and their shaped immune microenvironment highly facilitated the activation of downstream immune cells, including T cells, macrophages, and natural killer cells. FB-P30 was most similar to FB-E in terms of general transcriptome features, such as FB migration and proliferation, and aging-related characteristics. FB-D-gal showed the lowest expression levels of senescence-related genes. In comparisons with the single-cell RNA sequencing results, FB-E showed almost complete simulation of the transcriptional spectrum of FBs in elderly patients with atopic dermatitis, followed by FB-P30 and FB-UVB. FB-E and FB-P30showed higher similarity with the FBs in keloids. Each senescent FB model exhibited different characteristics. In addition to showing upregulated expression of natural senescence features, FB-UVB and FB-ATV showed high expression levels of senescence-related genes, including those involved in the SASP, and FB-P30showed the greatest similarity with FB-E. However, D-galactose-stimulated FBs did not clearly present aging characteristics.

Targeted partial reprogramming of age-associated cell states improves markers of health in mouse models of aging.

Aging is a complex multifactorial process associated with epigenome dysregulation, increased cellular senescence, and decreased rejuvenation capacity. Short-term cyclic expression of octamer-binding transcription factor 4 (Oct4), sex-determining region Y-box 2 (Sox2), Kruppel-like factor 4 (Klf4), and cellular myelocytomatosis oncogene (cMyc) (OSKM) in wild-type mice improves health but fails to distinguish cell states, posing risks to healthy cells. Here, we delivered a single dose of adeno-associated viruses (AAVs) harboring OSK under the control of the cyclin-dependent kinase inhibitor 2a (Cdkn2a) promoter to specifically partially reprogram aged and stressed cells in a mouse model of Hutchinson-Gilford progeria syndrome (HGPS). Mice showed reduced expression of proinflammatory cytokines and extended life spans upon aged cell-specific OSK expression. The bone marrow and spleen, in particular, showed pronounced gene expression changes, and partial reprogramming in aged HGPS mice led to a shift in the cellular composition of the hematopoietic stem cell compartment toward that of young mice. Administration of AAVs carrying Cdkn2a-OSK to naturally aged wild-type mice also delayed aging phenotypes and extended life spans without altering the incidence of tumor development. Furthermore, intradermal injection of AAVs carrying Cdkn2a-OSK led to improved wound healing in aged wild-type mice. Expression of CDKN2A-OSK in aging or stressed human primary fibroblasts led to reduced expression of inflammation-related genes but did not alter the expression of cell cycle-related genes. This targeted partial reprogramming approach may therefore facilitate the development of strategies to improve health and life span and enhance resilience in the elderly.

49912 The Effects of GHRH and its Analogue on Human Senescent Fibroblasts: Implications in Wound Healing in the Elderly

49912 The Effects of GHRH and its Analogue on Human Senescent Fibroblasts: Implications in Wound Healing in the Elderly

Co-Delivery of Valsartan and Metformin from a Thermosensitive Hydrogel-Nanoparticle System Promotes Collagen Production in Proliferating and Senescent Primary Human Dermal Fibroblasts.

Aging negatively impacts skin health, notably through the senescent cell phenotype, which reduces collagen production and leads to thinner, more fragile skin prone to injuries and chronic wounds. We designed a drug delivery system that addresses these age-related issues using a hybrid hydrogel-nanoparticle system that utilizes a poly(δ-valerolactone-co-lactide)-b-poly(ethylene-glycol)-b-poly(δ-valerolactone-co-lactide) (PVLA-PEG-PVLA) hydrogel. This hydrogel allows for the local, extended release of therapeutics targeting both proliferating and senescent cells. The PVLA-PEG-PVLA hydrogel entrapped valsartan, and metformin-loaded liposomes functionalized with a fibronectin-mimetic peptide, PR_b. Metformin acts as a senomorphic, reversing aspects of cellular senescence, and valsartan, an angiotensin receptor blocker, promotes collagen production. This combination treatment partially reversed the senescent phenotype and improved collagen production in senescent dermal fibroblasts from both young and old adults. Our codelivery hydrogel-nanoparticle system offers a promising treatment for improving age-related dermal pathologies.

Respiratory dysfunction in old mice could be related to inflammation and lung fibrosis induced by hyperphosphatemia.

With age, lungs undergo typical changes that lead to a deterioration of respiratory function. Our aim was to assess the role of age-associated hyperphosphatemia in these changes. We used C57BL6 mice to study an ageing model invivo and human lung fibroblasts were treated with a phosphate donor, beta-glycerophosphate (BGP), to explore mechanisms involved. Respiratory function was registered with a double chamber plethysmograph. Lung structure was analysed by different staining, phosphate and cytokines levels by colorimeric kits, expression of fibrosis, inflammation and ET-1 system by western blot or RT-PCR. Old mice showed hyperphosphatemia, along with lung fibrosis, loss of elastin, increased expression of pro-inflammatory cytokines and impaired respiratory function. BGP induced inflammation and fibrosis in fibroblasts through the activation and binding of NFkB to the MCP-1 or FN promoters. BGP increased ECE-1 expression by inducing NFkB binding to the ECE-1 promoter. QNZ, an NFkB inhibitor, blocked these effects. When ECE-1 was inhibited with phosphoramidon, BGP-induced inflammation and fibrosis were significantly reduced, suggesting a role for ET-1 in BGP-mediated effects.ET-1 produced effects similar to those of BGP, which were also dependent on NFkB. To study the pathophysiological relevance of hyperphosphatemia invivo, a low-P diet was administered to a group of old animals, showing an improvement in fibrosis, inflammation and respiratory function compared to old mice on a standard diet. These results suggest that age-related hyperphosphatemia induces inflammation, fibrosis, and impaired respiratory function in old mice; these effects appear to be mediated by ET-1 and NFkB activation.

Functional hyaluronic acid microneedles for skin photoaging based on collagen induction and oxidative stress regulation strategies

Photoaging holds remarkable importance for skin health and senescence. Ultraviolet (UV) irradiation results in the disruption of the extracellular matrix (ECM) microenvironment, the degradation of collagen, and the generation of oxidative stress. Traditional hyaluronic acid (HA) exhibits a diminished capacity to stimulate collagen regeneration, and hampered by its poor permeability as a macromolecule, ultimately resulting in constrained therapeutic outcomes for the treatment of photoaging. In this study, HA/PX was prepared by functional modification of HA with sulfonate-rich or phosphatidylcholine-rich polymers, which could complement the loss of ECM and ameliorate the senescence of human fibroblasts (HDFs) and hairless mouse models subjected to UVB-induced photoaging. The results indicate that HA/PX exhibits superior abilities in delaying cellular aging, promoting collagen regeneration, and resisting reactive oxygen species (ROS) compared to HA. Furthermore, HA/PX shows good biocompatibility both in vivo and in vitro, without causing allergic reactions or other adverse effects. We also demonstrated that the transdermal delivery of HA/PX via microneedle arrays (MNs) can significantly mitigate wrinkles and skin damage in photoaged nude mice, and achieve the treatment of skin photoaging by enhancing epidermal thickness, promoting collagen deposition, and reducing oxidative stress. Therefore, our research offers a novel possibility for future anti-aging therapeutic strategies.

Exosomes derived from hTERT-immortalized cells delay cellular senescence of human fibroblasts

hTERT gene therapies hold significant promise for treating age-related diseases. However, further research is required to address the challenges of delivery and ethical considerations. We hypothesized that exosomes derived from hTERT-immortalized cells could function similarly to hTERT gene therapies by maintaining telomere length and attenuating cellular senescence biomarkers. In this study, we overexpressed the hTERT gene in Human Foreskin Fibroblast-1 cells (HFF cells) to produce hTERT-immortalized HFF cells (hT-HFF cells). We then used exosomes derived from these hT-HFF cells to treat human fibroblasts, HFF cells. Our results demonstrated that these exosomes effectively attenuated biomarkers of cellular senescence in HFF cells. Furthermore, analysis revealed that hTERT mRNA was indeed packaged into the exosomes from hT-HFF cells. This mRNA was capable of elongating telomeres and delaying cellular senescence in HFF cells. Therefore, exosomes from hT-HFF cells show potential as a treatment for age-related diseases.

Addition of Polyphenols to Drugs: The Potential of Controlling "Inflammaging" and Fibrosis in Human Senescent Lung Fibroblasts In Vitro.

The combination of a polyphenol, quercetin, with dasatinib initiated clinical trials to evaluate the safety and efficacy of senolytics in idiopathic pulmonary fibrosis, a lung disease associated with the presence of senescent cells. Another approach to senotherapeutics consists of controlling inflammation related to cellular senescence or "inflammaging", which participates, among other processes, in establishing pulmonary fibrosis. We evaluate whether polyphenols such as caffeic acid, chlorogenic acid, epicatechin, gallic acid, quercetin, or resveratrol combined with different senotherapeutics such as metformin or rapamycin, and antifibrotic drugs such as nintedanib or pirfenidone, could present beneficial actions in an in vitro model of senescent MRC-5 lung fibroblasts. A senescent-associated secretory phenotype (SASP) was evaluated by the measurement of interleukin (IL)-6, IL-8, and IL-1β. The senescent-associated β-galactosidase (SA-β-gal) activity and cellular proliferation were assessed. Fibrosis was evaluated using a Picrosirius red assay and the gene expression of fibrosis-related genes. Epithelial-mesenchymal transition (EMT) was assayed in the A549 cell line exposed to Transforming Growth Factor (TGF)-β in vitro. The combination that demonstrated the best results was metformin and caffeic acid, by inhibiting IL-6 and IL-8 in senescent MRC-5 cells. Metformin and caffeic acid also restore cellular proliferation and reduce SA-β-gal activity during senescence induction. The collagen production by senescent MRC-5 cells was inhibited by epicatechin alone or combined with drugs. Epicatechin and nintedanib were able to control EMT in A549 cells. In conclusion, caffeic acid and epicatechin can potentially increase the effectiveness of senotherapeutic drugs in controlling lung diseases whose pathophysiological component is the presence of senescent cells and fibrosis.

Exosomes Released from Senescent Cells and Circulatory Exosomes Isolated from Human Plasma Reveal Aging-associated Proteomic and Lipid Signatures.

Senescence emerged as a significant mechanism of aging and age-related diseases, offering an attractive target for clinical interventions. Senescent cells release a senescence-associated secretory phenotype (SASP), including exosomes that may act as signal transducers between distal tissues, propagating secondary or bystander senescence and signaling throughout the body. However, the composition of exosome SASP remains underexplored, presenting an opportunity for novel unbiased discovery. Here, we present a detailed proteomic and lipidomic analysis of exosome SASP using mass spectrometry from human plasma from young and older individuals and from tissue culture of senescent primary human lung fibroblasts. We identified ~1,300 exosome proteins released by senescent fibroblasts induced by three different senescence inducers causing most exosome proteins to be differentially regulated with senescence. In parallel, a human plasma cohort from young and old individuals revealed over 1,350 exosome proteins and 171 plasma exosome proteins were regulated when comparing old vs young individuals. Of the age-regulated plasma exosome proteins, we observed 52 exosome SASP factors that were also regulated in exosomes from the senescent fibroblasts, including serine protease inhibitors (SERPINs), Prothrombin, Coagulation factor V, Plasminogen, and Reelin. In addition, 247 lipids were identified with high confidence in all exosome samples. Following the senescence inducers, a majority of the identified phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin species increased significantly indicating cellular membrane changes. The most notable categories of significantly changed proteins were related to extracellular matrix remodeling and inflammation, both potentially detrimental pathways that can damage surrounding tissues and even induce secondary or bystander senescence. Our findings reveal mechanistic insights and potential senescence biomarkers, enabling a better approach to surveilling the senescence burden in the aging population and offering promising therapeutic targets for interventions.

Senescent glia link mitochondrial dysfunction and lipid accumulation

Senescence is a cellular state linked to ageing and age-onset disease across many mammalian species1,2. Acutely, senescent cells promote wound healing3,4 and prevent tumour formation5; but they are also pro-inflammatory, thus chronically exacerbate tissue decline. Whereas senescent cells are active targets for anti-ageing therapy6–11, why these cells form in vivo, how they affect tissue ageing and the effect of their elimination remain unclear12,13. Here we identify naturally occurring senescent glia in ageing Drosophila brains and decipher their origin and influence. Using Activator protein 1 (AP1) activity to screen for senescence14,15, we determine that senescent glia can appear in response to neuronal mitochondrial dysfunction. In turn, senescent glia promote lipid accumulation in non-senescent glia; similar effects are seen in senescent human fibroblasts in culture. Targeting AP1 activity in senescent glia mitigates senescence biomarkers, extends fly lifespan and health span, and prevents lipid accumulation. However, these benefits come at the cost of increased oxidative damage in the brain, and neuronal mitochondrial function remains poor. Altogether, our results map the trajectory of naturally occurring senescent glia in vivo and indicate that these cells link key ageing phenomena: mitochondrial dysfunction and lipid accumulation.

P03 Anti-ageing effects of oestetrol on the skin: protection from cellular senescence and restoration of dermal architecture

Abstract Introduction and aims Skin is a major endocrine organ, undergoing profound structural and functional alterations in response to hormonal changes. Postmenopause, the rapid decline in circulating 17β-oestradiol (E2) leads to skin ageing, while exogenous E2 administration restores skin function, promoting the regenerative capacity of aged skin. Nevertheless, the risk of off-target effects limits the clinical use of E2 for skin indications. Oestetrol (E4), a natural oestrogenic steroid currently undergoing clinical trials as a hormone replacement therapy, presents a favourable safety profile and could be a suitable alternative to E2. However, as no studies have investigated the effects E4 on skin ageing, the aim of this study was to evaluate the potential of E4 to overcome effects of skin ageing. Methods Micro-osmotic pumps were subcutaneously implanted into 14-month-old female mice, allowing sustained systemic delivery of E2 or E4 over 3 weeks. Reversal of skin ageing was assessed via noninvasive skin parameter testing, histological analysis and biomechanics. Aged human dermal fibroblasts were treated with E4 to measure effects on the extracellular matrix deposition (ECM) in vitro. Finally, human keratinocytes were treated with E4 prior to inducing senescence to assess potential protective effects against skin ageing. Readouts included senescence-associated beta galactosidase staining, immunofluorescence and quantitative polymerase chain reaction. Results Systemic administration of E4 reversed multiple characteristics of skin ageing. E4 treatment significantly increased skin hydration, stimulated collagen deposition and elevated elastic fibre synthesis. These structural changes improved the biomechanical function of skin, increasing breaking strength and elasticity. In vitro studies confirmed direct anti-ageing activity on multiple cell types. E4 substantially increased ECM deposition in aged human dermal fibroblasts and protected against senescence induction in human keratinocytes. Conclusions Collectively, these preclinical data demonstrate significant beneficial effects of E4 on aged skin, strongly supporting future clinical evaluation of E4 to alleviate the effects of skin ageing following menopause.

Combined dasatinib and quercetin treatment contributes to skin rejuvenation through selective elimination of senescent cells in vitro and in vivo.

The skin's protective functions are compromised over time by both endogenous and exogenous aging. Senescence is well-documented in skin phenotypes, such as wrinkling and sagging, a consequence of the senescence-associated secretory phenotype (SASP) that involves the accumulation of senescent fibroblasts, chronic inflammation, and collagen remodeling. Although therapeutic approaches for eliminating senescent cells from the skin are available, their efficacy remains unclear. Accordingly, we aimed to examine the effects of dasatinib in combination with quercetin (D + Q) on senescent human skin fibroblasts and aging human skin. Senescence was induced in human dermal fibroblasts (HDFs) using approaches such as long-term passaging, ionizing radiation, and doxorubicin treatment. The generated senescent cells were treated with D + Q or vehicle. Additionally, a mouse-human chimera model was generated by subcutaneously transplanting whole-skin grafts of aged individuals onto nude mice. Mouse models were administered D + Q or vehicle by oral gavage for 30 days. Subsequently, skin samples were harvested and stained for senescence-associated beta-galactosidase. Senescence-associated markers were assessed by western blotting, reverse transcription-quantitative PCR and histological analyses. Herein, D + Q selectively eliminated senescent HDFs in all cellular models of induced senescence. Additionally, D + Q-treated aged human skin grafts exhibited increased collagen density and suppression of the SASP compared with control grafts. No adverse events were observed during the study period. Collectively, D + Q could ameliorate skin aging through selective elimination of senescent dermal fibroblasts and suppression of the SASP. Our findings suggest that D + Q could be developed as an effective therapeutic approach for combating skin aging.