Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin

Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative‐senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age‐related phenotypic and functional alteration of fibroblasts from ‘young’ (<35 years) and ‘old’ (>50 years) donors. Our results brought evidence of the senescent status of ‘old’ fibroblasts by senescence associated β‐galactosidase (SA‐βgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in ‘old’ fibroblasts as well as a subset of upregulated genes involved in senescence features. In ‘old’ fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFβ. Old fibroblasts were also more sensitive to oxidative stress than ‘young’ ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of ‘old’ fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.     

SASP‐induced macrophage dysfunction may contribute to accelerated senescent fibroblast accumulation in the dermis

Recently, increasing attention has been paid to senescence‐associated secretory phenotype (SASP), a phenomenon that senescent cells secrete molecules such as inflammatory cytokines and matrix metalloproteinases (MMPs), due to its noxious effects on the surrounding tissue. Senescent cells in the blood and liver are known to be properly depleted by macrophages. In the dermis, accumulation of senescent cells has been reported and is thought to be involved with skin ageing. In this study, to elucidate the clearance mechanism of senescent cells in the dermis, we focused on macrophage functions. Our co‐culture experiments of senescent fibroblasts and macrophages revealed a two‐step clearance mechanism: first, TNF‐α secreted from macrophages induces apoptosis in senescent fibroblasts, and then, dead cells are phagocytosed by macrophages. Furthermore, it was suggested that SASP factors suppress both of the two steps of the senescent cell clearance by macrophages. From these findings, normally senescent cells in the dermis are thought to be removed by macrophages, but when senescent cells are excessively accumulated owing to oxidative stress, ultraviolet (UV) ray or other reasons, SASP was suggested to suppress the macrophage‐dependent clearance functions and thereby cause further accumulation of senescent cells.     

Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture

The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high‐passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high‐passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti‐skin‐ageing strategies.     

High TMEM45A expression is correlated to epidermal keratinization

TMEM45A (DERP7, DNAPTP4 or FLJ10134) gene, belonging to the TMEM family encoding predicted transmembrane proteins, is highly expressed in epidermal keratinocytes. To investigate the potential involvement of TMEM45A during the differentiation and keratinization processes, its expression has been characterized in normal human keratinocytes and the protein subcellular localization has been studied in this cell type, both in vitro and in vivo. TMEM45A expression is upregulated with differentiation, either induced by cultured keratinocyte confluence or enhanced Ca²⁺ concentration in medium. In vivo, TMEM45A mRNA and protein are mostly found in the granular layer of the epidermis. TMEM45A expression is linked to keratinization, as accumulation of the protein is detected in native and reconstructed epidermis as well as in thymic Hassal bodies, but not in non‐keratinized stratified epithelia. At the subcellular level, co‐detection with ER and Golgi markers reveals that TM protein 45A is associated with the Golgi apparatus and more specifically with the trans‐Golgi/trans‐Golgi network in vitro and in granular layer in vivo. The protein is neither related to lysosomes nor transported within corneodesmosin‐containing lamellar bodies. These data demonstrate a strong correlation between TMEM45A expression and epidermal keratinization, indicating the relevance of this gene in this process.     

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells

Skin is one of the most commonly studied tissues for microcirculation research owing to its close correlation of cutaneous vascular function, ageing and age‐related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and ageing in the vascular environment of the skin, we performed a proteomic analysis of plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Among identified proteins, paraoxonase 1 (PON1) was selected in this study. To elucidate the role of PON1 on skin ageing and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked‐down by small interfering RNA (siRNA) targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence‐associated β‐galactosidase stained cells and enlarged and flattened cell morphology. At 48 h post‐transfection, the protein expression of p16 in PON1 siRNA‐treated HDMECs was higher than that in scrambled siRNA‐treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age‐related candidate biomarkers, were decreased by PON1 knock‐down in HDMECs. In conclusion, these results suggest that PON1 functions as an ageing‐related protein and plays an important role in the cellular senescence of HDMECs.     

The natural phytochemical trans‐communic acid inhibits cellular senescence and pigmentation through FoxO3a activation

Ageing is characterized by the accumulation of chronic and irreversible oxidative damage, chronic inflammation and organ dysfunction. To attenuate these ageing‐related changes, various natural phytochemicals are often applied. Trans‐communic acid (TCA), an active component of brown pine leaf extract, has antimicrobial and cancer chemopreventive activity and inhibits ultraviolet B (UVB)‐induced MMP‐1 expression. To determine whether the phytochemical TCA could affect the lifespan of an ageing model, Caenorhabditis elegans prevent ageing‐related phenotypes of the skin. Caenorhabditis elegans (C. elegans) wild‐type N2 and mutant strains were used in this study to explore the lifespan extension effect of TCA and its mechanism. We estimated lipofuscin accumulation and melanin levels, which are closely associated with skin senescence. Moreover, we explored the mechanism of action associated with ageing attenuation. We performed oxidative stress resistance and thermotolerance assays in C. elegans and surface plasmon resonance analysis of TCA binding with the forkhead box‐O3a (FoxO3a) protein. TCA, which is the active component in Korean red pine (Pinus densiflora), attenuated ageing‐related changes in skin cells. TCA lowered lipofuscin accumulation in fibroblasts and decreased melanin levels in melanocytes. These protective effects were mediated by activation of the representative longevity gene FoxO3a, which was induced by direct binding with TCA. Interestingly, TCA extended the lifespan of C. elegans, although it did not affect stress resistance, oxidative stress or thermotolerance. These results strongly suggest that TCA prevents the senescent phenotype of model organisms and exhibits beneficial effects on ageing‐related skin phenotypes through direct FoxO3a activation.     

Senescent fibroblasts in melasma pathophysiology

It has been proposed that melasma is a photoageing skin disorder. The photoaged fibroblasts have been suggested as an important source of melanogenic factors which are involved in the regulation of pigmentation. To investigate whether melasma includes senescent cells, lesional and perilesional normal skin from 38 melasma patients was assessed using a cell senescence marker, p16^INK4A. The results showed that lesional dermal skin had more p16^INK4A‐positive senescent cells than perilesional skin. The impact of senescent fibroblasts was further investigated in a pilot study using radiofrequency (RF) intervention for melasma. It showed that the RF therapy decreased the number of senescent cells with increased expression of procollagen‐1, which were associated with reduced epidermal pigmentation. This leads us to the speculation that senescent fibroblasts may contribute to drive melasma and might be considered as a potential therapeutic target.     

GPHR-dependent functions of the Golgi apparatus are essential for the formation of lamellar granules and the skin barrier

The lumen of the Golgi apparatus is regulated to be weakly acidic, which is critical for its functions. The Golgi pH regulator (GPHR) is an anion channel essential for normal acidification of the Golgi apparatus, and is therefore required for its functions. The Golgi apparatus has been thought to be the origin of lamellar granules in the skin. To study the functional role(s) of GPHR in the skin, we established keratinocyte-specific GPHR-knockout mice using the Cre-loxP system. These mutant mice exhibited hypopigmented skin, hair loss, and scaliness. Histological examination of GPHR-knockout mice showed ballooning of the basal cells and follicular dysplasia. In addition, inflammatory cells were seen in the dermis. The expression of trans-Golgi network 46, a marker for lamellar bodies, and kallikrein 7, a protein within lamellar bodies, is diminished in GPHR-knockout mouse skin. Examination by electron microscopy revealed that keratinocytes produced aberrant lamellar bodies. The transepidermal water loss of these knockout mice was increased compared with wild-type mice. Moreover, expression of cathelicidin-related antimicrobial peptide (CRAMP) in the skin was diminished. These results suggest that GPHR is essential for the homeostasis of the epidermis including the formation of lamellar bodies and for the barrier function.     

Particulate matter induces pro‐inflammatory cytokines via phosphorylation of p38 MAPK possibly leading to dermal inflammaging

Particulate matter (PM) is known to have harmful effects on human health. Epidemiological studies have suggested that PM exposure is related to skin diseases and extrinsic skin ageing. However, the mechanisms by which PM affects skin are unclear. The aim of this study was to investigate the mechanism of action of PMs on epidermal inflammation and skin ageing using a co‐culture of human keratinocytes (HaCaT) and fibroblasts (HDF). SRM 1648a (pmA) and 1649b (pmB), which mainly comprise heavy metals and polycyclic aromatic hydrocarbons, respectively, were used as reference PMs. Cytotoxic effects, activation of AhR, phosphorylation of p38 kinase and ROS generation were examined in PM‐treated HaCaT cells. The phosphorylation of p38 MAPK induced by PMs was shown to be critically important for the increases in IL‐1α and IL‐1β expression. Moreover, the mRNA and protein expression levels of MMP1 and COX2 were markedly increased in HDF cells co‐cultured with PM‐treated HaCaT cells. In conclusion, PMs induce the expression of pro‐inflammatory cytokines in keratinocytes via the p38 MAPK pathway, and these interleukins increase the expression of MMP1 and COX2 in HDF cells. These results suggest that PMs trigger skin ageing via p38 MAPK activation and interleukin secretion in epidermal keratinocytes.     

Heat-labile glucose-6-phosphate dehydrogenase in cultured fibroblasts from patients with De Sanctis-Cacchione syndrome

Summary The normal senescent fibroblasts in culture accumulate a significantly high proportion of altered enzymes, and the alterations are considered to be the manifestation of ageing in molecular terms. To detect the possible molecular alterations in patients with De Sanctis-Cacchione syndrome, the severest form of xeroderma pigmentosum, in which repair processes to UV light-damaged DNA are defective and the neurologic abnormalities are considered to reflect accelerated ageing, we studied the heat stability of glucose-6-phosphate dehydrogenase (G6PD) in crude extracts of cultured skin fibroblasts. Three patients with the syndrome were the center of our investigation. Even at early passage in culture the heat-labile portion of G6PD was increased in the cells from patients in comparison to normal controls.The life span of the cells in culture from patients was not reduced below normal age-matched controls, and no appreciable senescent appearance was observed. The increase in the heat-labile portion of G6PD from cells of De Sanctis-Cacchione syndrome patients to reflect that defective repair of DNA damage occurs, rather than being a direct result of ageing of cultured cells.     

Differential roles for Chk1 and FANCD2 in ATR-mediated signalling for psoralen photoactivation-induced senescence

Cellular senescence is a stress-inducible, naturally irreversible cell cycle arrest, which is likely linked with ageing. Premature ageing of the skin is a prominent side effect of psoralen photoactivation, which is used for the treatment of various skin disorders. Previously, we have shown that DNA interstrand crosslink formation by photoactivated psoralens induces a senescent phenotype in primary fibroblasts that is mediated by Ataxia telangiectasia-mutated and Rad3-related (ATR) kinase. Checkpoint kinase 1 (Chk1) initiates cell cycle checkpoints, and FANCD2 is known to be involved in DNA damage-induced S-phase arrest and crosslink repair. In this study, we examined a role for Chk1 and FANCD2 as downstream effectors of ATR in senescence signalling. We demonstrate that Chk1 and FANCD2 are long-lastingly activated after psoralen photoactivation. Separate and combined reduction in Chk1 and FANCD2 expression by small interfering RNA (siRNA) preceding irradiation partly prevented the initiation of the senescence-like phenotype, whereas siRNA (Chk1 and FANCD2) transfection of senesced fibroblasts released cells from growth arrest. We observed that Chk1 and FANCD2 signal equally and additively for senescence induction, while Chk1 is predominantly responsible for maintaining persistent cell cycle arrest. In conclusion, Chk1 and FANCD2 function downstream of ATR in a non-redundant manner for the establishment and maintenance of psoralen photoactivation-induced senescence.     

The effect of moesin overexpression on ageing of human dermal microvascular endothelial cells

Abstract:  Senescence of microvascular endothelial cells is known to play an important role in the pathophysiology of vascular diseases related to ageing, but the accurate mechanism or related genes are not known. Moesin, a cytoskeletal protein and the most potent candidate as an ageing-related protein, showed obvious changes in expression when compared before and after ageing. In this study, a lentivirus was used to overexpress moesin in endothelial cells. The expression of cell cycle mediators such as p16, cyclin D1 and cdk4, which can be the markers of ageing, was compared by RNA and was shown to be suppressed in moesin overexpressed endothelial cells. In conclusion, it can be said that the expression of moesin delays senescence of human dermal microvascular endothelial cells and this fundamental discovery can be used as a basis for understanding the mechanism of ageing and age-related diseases.     

Oxytocin alleviates cellular senescence through oxytocin receptor-mediated extracellular signal-regulated kinase/Nrf2 signalling

Intrinsic skin aging is skin aging affected by natural factors within the body, such as hormones, rather than by external factors like UV light or smoking. Skin ageing causes the appearance of wrinkles, skin sagging and reduced elasticity. Like elsewhere in the body, skin ageing involves an increase in the proportion of cells undergoing cellular senescence (a type of deterioration due to age). Senescent skin cells called dermal fibroblasts have several characteristics that are different to cells that are not senescent, including having what is known as a senescence-associated secretory phenotype (SASP). Oxytocin (OT) is a neuropeptide hormone that has many beneficial effects in the body, including protection against age-related disorders. However, less is known about the role of OT in intrinsic skin aging. The authors looked into the role of oxytocin in prevention against senescence in skin cells called normal human dermal fibroblasts (NHDFs), taken from female donors of different ages. They found that OT suppressed SASP-induced senescence in NHDFs derived from young but not old female donors. The authors were also able to learn more about the processes within the body by which OT suppresses SASP and why it affects younger women differently to older women. Their results demonstrate that oxytocin may be effective in the prevention of skin aging.     

A three‐dimensional skin equivalent reflecting some aspects of in vivo aged skin

Human skin undergoes morphological, biochemical and functional modifications during the ageing process. This study was designed to produce a 3‐dimensional (3D) skin equivalent in vitro reflecting some aspects of in vivo aged skin. Reconstructed skin was generated by co‐culturing skin fibroblasts and keratinocytes on a collagen–glycosaminoglycan–chitosan scaffold, and ageing was induced by the exposition of fibroblasts to Mitomycin‐C (MMC). Recently published data showed that MMC treatment resulted in a drug‐induced accelerated senescence (DIAS) in human dermal fibroblast cultures. Next to established ageing markers, histological changes were analysed in comparison with in vivo aged skin. In aged epidermis, the filaggrin expression is reduced in vivo and in vitro. Furthermore, in dermal tissue, the amount of elastin and collagen is lowered in aged skin in vivo as well as after the treatment of 3D skin equivalents with MMC in vitro. Our results show histological signs and some aspects of ageing in a 3D skin equivalent in vitro, which mimics aged skin in vivo.     

Reduction in DNA methyltransferases and alteration of DNA methylation pattern associate with mouse skin ageing

Understanding molecular mechanisms of skin ageing is critical for developing effective anti‐ageing strategies. Recently, it has been suggested that epigenetics maybe be involved in tissue ageing and age‐related diseases; however, the evidence regarding skin ageing has been very limited. We ran a pilot study in mouse skin to test whether DNA methyltransferases (Dnmts), DNA demethylases such as ten‐eleven translocation enzymes (Tets) and DNA methylation of gene promoters change with age by quantitative RT‐PCR and methylated DNA immunoprecipitation (MeDIP)‐chip. We discovered that the expression of Dnmt3a, Dnmt3b and Tet2 declines significantly with skin ageing. The genome‐wide DNA methylation analysis indicates that both hypermethylation and hypomethylation in promoters of genes are taken place. Functional category of those genes suggests that inhibition of cell proliferation and activation of immune response are important adaptations likely induced by skin ageing. These findings shed new light on epigenetic regulation of skin ageing.     

Human mesenchymal stem cells may be involved in keloid pathogenesis

Background The pathogenesis of keloid is poorly understood. Although vigorous investigations have attempted to elucidate the mechanisms or causative factors of keloid, there are little data on why keloids are very intractable and recur easily in each patient. Methods In an attempt to analyze the possible interaction between human mesenchymal stem cells and keloid‐derived fibroblasts, the dual‐chamber cell‐migration assay, cell proliferation, ultrastructural morphology, and Western blot analysis were used to investigate the production of the extracellular matrices of the coculture. Results Cell proliferation was not significantly different between keloid‐derived fibroblasts and normal dermal fibroblasts during a 4‐day observation period. There was a significant cell migration of human mesenchymal stem cells when keloid‐derived fibroblasts were placed in the bottom chamber, compared to when normal dermal fibroblasts were placed in the same way in 8‐µm diameter pore membranes (190.6 ± 51.45 and 32.0 ± 6.20 cells/field, respectively, P < 0.01). With 3‐µm diameter pores, the human mesenchymal stem cells migrated in the pores only when the keloid‐derived fibroblasts were placed in the bottom chambers (6.4 ± 3.84 cells/field). Monolayer coculture of human mesenchymal stem cells and keloid‐derived fibroblasts demonstrated further functional differentiation, such as collagen secretion and abundant rough endoplasmic reticulum. Western blot analysis of the cells in the modified dual‐chamber culture demonstrated most significantly abundant fibronectin expression when the human mesenchymal stem cells contained keloid fibroblasts. Conclusion The results of this study may indicate that human mesenchymal stem cells participate and recruit in keloid pathogenesis by differentiating themselves toward keloid recalcitrant formation and progression.     

Metformin alleviates ageing cellular phenotypes in Hutchinson–Gilford progeria syndrome dermal fibroblasts

Metformin is a popular antidiabetic biguanide, which has been considered as a candidate drug for cancer treatment and ageing prevention. Hutchinson–Gilford progeria syndrome (HGPS) is a devastating disease characterized by premature ageing and severe age‐associated complications leading to death. The effects of metformin on HGPS dermal fibroblasts remain largely undefined. In this study, we investigated whether metformin could exert a beneficial effect on nuclear abnormalities and delay senescence in fibroblasts derived from HGPS patients. Metformin treatment partially restored normal nuclear phenotypes, delayed senescence, activated the phosphorylation of AMP‐activated protein kinase and decreased reactive oxygen species formation in HGPS dermal fibroblasts. Interestingly, metformin reduced the number of phosphorylated histone variant H2AX‐positive DNA damage foci and suppressed progerin protein expression, compared to the control. Furthermore, metformin‐supplemented aged mice showed higher splenocyte proliferation and mRNA expression of the antioxidant enzyme, superoxide dismutase 2 than the control mice. Collectively, our results show that metformin treatment alleviates the nuclear defects and premature ageing phenotypes in HGPS fibroblasts. Thus, metformin can be considered a promising therapeutic approach for life extension in HGPS.