Senescent Dermal Fibroblasts Research Articles

Cryptomphalus aspersa mollusc eggs extract promotes migration and prevents cutaneous ageing in keratinocytes and dermal fibroblasts in vitro.

The search of substances that minimize cutaneous ageing has increased in the last few years. Previous studies have described the regenerative properties of the secretion of the mollusc Cryptomphalus aspersa (C.aspersa) when applied topically. We evaluate the in vitro effects of a new product derived from the eggs of C.aspersa, IFC-CAF, on cell proliferation, migration, distribution of cytoskeletal proteins, production of extracellular components as well as its ability to prevent cutaneous ageing because of intrinsic or extrinsic factors (exposure to UVB) by determination of ageing markers. We have used the human keratinocyte cell line (HaCaT cells), primary dermal fibroblasts (HDF) and senescent dermal fibroblasts (SHDF). The effects of the compound on cell proliferation and on the cell cycle were determined by the MTT colorimetric assay, estimation of total protein and/or trypan blue test and by flow cytometry, respectively. We also studied cell migration using the wound-healing migration assay, whereas ELISA assays, Western Blot and immunofluorescence microscopy were carried out to test the expression of proteins related to cytoskeleton, extracellular matrix and with ageing. We have found that IFC-CAF does not promote proliferation but induces migration of HaCaT, HDF and SHDF in a time- and dose-dependent manner; a better organization of cytoskeletal proteins (F-actin and vimentin) and promotes the production of extracellular components (fibronectin, collagen 1 and MMPs) and the adhesion to cell-substrate vinculin protein. IFC-CAF also prevents cutaneous ageing. The treatment decreases the expression of the ageing-related markers b-Gal, p53 and p16INK4 in SDDF cells, and improves cell survival after UVB irradiation and nuclear repair in HaCaT cells. IFC-CAF has regenerative properties and protects against ageing factors being, therefore, a potential therapeutic agent for treating or preventing skin ageing.

Senescent Fibroblasts Enhance Early Skin Carcinogenic Events via a Paracrine MMP-PAR-1 Axis

The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.

Fibroblast Senescence and Squamous Cell Carcinoma: How Wounding Therapies Could Be Protective

Squamous cell carcinoma (SCC), which has one of the highest incidences of all cancers in the United States, is an age-dependent disease, with the majority of these cancers diagnosed in people age 70 and older. Recent findings have led to a new hypothesis on the pathogenesis of SCC. To evaluate the potential of preventive therapies to reduce the incidence of SCC in at-risk geriatric patients. Survey of current literature on wounding therapies to prevent SCCs. This new hypothesis of SCC photocarcinogenesis states that senescent fibroblasts accumulate in the dermis, resulting in a reduction in dermal insulin-like growth factor-1 (IGF-1) expression. This lack of IGF-1 expression sensitizes epidermal keratinocytes to fail to suppress ultraviolet light B (UVB)-induced mutations, leading to increased proclivity to photocarcinogenesis. Recent evidence suggests that dermal wounding therapies, specifically dermabrasion and fractionated laser resurfacing, can decrease the proportion of senescent dermal fibroblasts, increase dermal IGF-1 expression, and correct the inappropriate UVB response found in geriatric skin, protecting geriatric keratinocytes from UVB-induced SCC initiation. In this review, we will discuss the translation of pioneering basic science results implicating commonly used dermal fibroblast rejuvenation procedures as preventative treatments for SCC.

Elevated miR-34c-5p Mediates Dermal Fibroblast Senescence by Ultraviolet Irradiation

Previous studies showed that several miRNAs can regulate pathways involved in UVB-induced premature senescence and response to ultraviolet irradiation. It has also been reported that miR-34c-5p may be involved in senescence-related mechanisms. We propose that miR-34c-5p may play a crucial role in senescence of normal human primary dermal fibroblasts. Here, we explored the roles of miR-34c-5p in UVB-induced premature senescence on dermal fibroblasts. MiR-34c-5p expression was increased in dermal fibroblasts after repeated subcytotoxic UVB treatments. Underexpression of miR-34c-5p in dermal fibroblasts led to a marked delay of many senescent phenotypes induced by repeated UVB treatments. Furthermore, underexpression of miR-34c-5p in dermal fibroblasts can antagonize the alteration of G1-arrested fibroblasts. Moreover, E2F3, which can inactivate p53 pathway and play a role in cell cycle progression, is a down-stream target of miR-34c-5p. Forced down-expression of miR-34c-5p decreased the expression of UVB-SIPS induced P21 and P53 at both mRNA and protein levels. Our data demonstrated that down-regulation of miR-34c-5p can protect human primary dermal fibroblasts from UVB-induced premature senescence via regulations of some senescence-related molecules.

Cysteine‐rich protein 61 (CCN1) mediates replicative senescence‐associated aberrant collagen homeostasis in human skin fibroblasts

Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.

Age-Associated Decrease in Proteasome Content and Activities in Human Dermal Fibroblasts: Restoration of Normal Level of Proteasome Subunits Reduces Aging Markers in Fibroblasts From Elderly Persons

We measured proteasome activities and the levels of proteasome subunits in dermal fibroblasts from individuals aged 20-82 years. Proteasome activities changed with age in a biphasic manner, decreasing significantly up to 50 years of age and showing no significant change between 50 and 78 years of age. Similarly, proteasome activities in replicatively senescent dermal fibroblasts showed a passage-dependent biphasic change. We confirmed that the decreases in proteasome activities were accompanied by the accumulation of oxidized and ubiquitinated proteins. The decline in proteasome activities in aging fibroblasts was associated with a decrease in the expression of proteasome subunits. We found that the restoration of the normal level of proteasome catalytic subunits, using a lentivirus gene-delivery system, decreased the severity of the aging markers in dermal fibroblasts from elderly donors. These findings suggest that proteasome malfunction may contribute to the aging process in human skin and that the maintenance of normal proteasome activities could delay skin aging.

Melanocortin receptors in fibroblastic cell types of the skin –in vitro and in vivo expression and functional relevance

In contrast to the well‐established role of melanocortins on melanocyte function, the expression and relevance of melanocortin receptors (MC‐Rs) in skin fibroblasts is incompletely understood. We recently showed that human dermal fibroblasts derived from foreskin express functional MC‐1Rs which mediate an inhibitory action of α‐melanocyte‐stimulating hormone (α‐MSH) on collagen metabolism (Böhm et al., J Biol Chem 2004, in press). Here, we show by RT‐PCR, immunofluorescence, immunohistochemistry and immune electron microscopy that MC‐1R expression is conserved in vitro and in vivo in additional human fibroblastic cell types of the skin including adult dermal fibroblasts, fibrosarcoma cells, connective tissue sheath fibroblasts and dermal papilla cells of the hair follicle. In vitro expression of MC‐1R declines as a matter of cellular senescence in dermal fibroblasts. Interestingly, α‐MSH inhibits the inductive effect of interferon‐γ, an important proinflammatory mediator in inflammatory and fibrotic skin diseases, on expression of adhesion molecules such as intercellular adhesion molecule‐1 in human dermal fibroblasts. These data point towards an additional biological role of α‐MSH as a modulator of inflammatory reactions in the connective tissue compartment of the skin.

Fibroblast clones from patients with Hutchinson–Gilford progeria can senesce despite the presence of telomerase

Hutchinson–Gilford progeria (HGP) is a genetic disorder in which individuals prematurely display features of ageing. Mutations in LMNA (lamin A) have recently been shown to underlie HGP, although how such mutations lead to the complex phenotype seen in the disease remains unclear. HGP is often associated with the premature replicative senescence of dermal fibroblasts. Normally dermal fibroblast senescence is initiated by erosion of chromosomal ends (telomeres) resulting from sustained cell division. Since ectopic expression of telomerase reproducibly immortalises human dermal fibroblasts, it is of interest to determine whether HGP fibroblasts immortalise via the same route, and at the same frequency. Three strains of HGP fibroblasts (AGO6917A, AGO6297B and AGO8466) were infected with a retroviral vector expressing the catalytic subunit of telomerase (hTERT). Here we report that fibroblast clones derived from HGP donors frequently fail to immortalise with telomerase. Of the 15 independently isolated clones from the three donors, five failed to immortalise despite the restoration of telomerase activity and the stabilisation of telomere length. In contrast, out of four clones isolated from a culture of hTERT transduced control fibroblasts, no failures to immortalise were detected. This suggests a novel cellular phenotype in HGP, one whereby the HGP mutation confers resistance to ‘telomerisation’.

An analysis of replicative senescence in dermal fibroblasts derived from chronic leg wounds predicts that telomerase therapy would fail to reverse their disease-specific cellular and proteolytic phenotype

The accumulation of senescent fibroblasts within tissues has been suggested to play an important role in mediating impaired dermal wound healing, which is a major clinical problem in the aged population. The concept that replicative senescence in wound fibroblasts results in reduced proliferation and the failure of refractory wounds to respond to treatment has therefore been proposed. However, in the chronic wounds of aged patients the precise relationship between the observed alteration in cellular responses with aging and replicative senescence remains to be determined. Using assays to assess cellular proliferation, senescence-associated staining β-galactosidase, telomere length, and extracellular matrix reorganizational ability, chronic wound fibroblasts demonstrated no evidence of senescence. Furthermore, analysis of in vitro senesced fibroblasts demonstrated cellular responses that were distinct and, in many cases, diametrically opposed from those exhibited by chronic wound fibroblasts. Forced expression of telomerase within senescent fibroblasts reversed the senescent cellular phenotype, inhibiting extracellular matrix reorganizational ability, attachment, and matrix metalloproteinase production and thus produced cells with impaired key wound healing properties. It would appear therefore that the distinct phenotype of chronic wound fibroblasts is not simply due to the aging process, mediated through replicative senescence, but instead reflects disease-specific cellular alterations of the fibroblasts themselves.

Senescence and the healing rates of venous ulcers

Objective: Premature cellular senescence has been linked to venous hypertension and may contribute to delayed healing of venous ulcers. We hypothesized that the percentage of senescent cells in in vitro populations of fibroblasts isolated from venous ulcers is directly related to the clinical time-to-healing. Methods: Biopsy specimens were obtained from ulcer margins and unaffected dermal tissue of the ipsilateral thigh of seven patients with active venous ulcers. Using explant culture techniques, we obtained populations of wound fibroblasts and normal fibroblasts. The percentage of senescence in these cell populations was determined with X-Gal (5-bromo-4-chloro-3-indolyl β-D-galactoside), which was used as a stain for B-galactosidase, a biomarker for senescent dermal fibroblasts. The X-Gal stain is a peroxidase stain for B-galactosidase. All patients in the study were treated with compression dressings. On a weekly basis, digital images were taken until ulcers healed. Planimetric healing rates were calculated from these images, and an overall time-to-healing was recorded. All cytologic investigations were performed on first passage cells. Results: The average starting ulcer size was 4.2 cm 2. Five of the data points represented healed ulcers. The two remaining patients withdrew from the study to pursue other therapies after having been treated with compression dressings for a long time. Linear regression analysis of healed ulcers identified a relationship between percent of senescence and time-to-healing, which was statistically significant ( R 2 = 0.81, P = .037). High percentages of senescent cells also had a correlation with slowed planimetric healing, which was not statistically significant. Conclusions: This study demonstrates a clinical correlation between quantitative in vitro senescence and time-to-healing. A percentage of senescence that is greater than 15% in populations of cells isolated from venous ulcers may identify a “difficult to heal” ulcer. There is no good clinical indicator for determining the likelihood of ulcer healing, but these results indicate that senescence percentage may have potential in this regard. (J Vasc Surg 2001;33:1206-11.)

Effect of Cellular Senescence and Retinoic Acid on the Expression of Cellular Retinoic Acid Binding Proteins in Skin Fibroblasts

Retinoic acid (RA) has significant effects on a variety of cellular processes, including growth and differentiation. Retinoic acid has also been implicated as a major morphogen during embryogenesis. In the skin, both the epidermis and the dermis are extremely responsive to the effects of retinoids; however, the molecular mechanisms through which retinoids act in this tissue remain poorly understood. Two classes of proteins play roles in mediating the biological effects of retinoic acid. The nuclear receptors for retinoic acid are of two types, the initially described RARs, and the RXRs. Each of these nuclear receptor families has multiple isoforms and can function as a ligand-inducible transcription enhancing factor. A second class of receptor proteins exists for retinoic acid that are found in the cytoplasm, the cellular retinoic acid binding proteins (CRABPs). These proteins play a role in the binding, transport, and metabolism of retinoic acid. In previous studies, we have demonstrated that retinoic acid induces the expression of RARβ and RARγ in human dermal fibroblasts. Moreover, we have also shown the selective transcriptional up-regulation of the RARβ2 isoform in senescent dermal fibroblasts and senescent human mammary epithelial cells. In order to further define molecules important in regulating the response of senescent dermal fibroblasts to retinoids, we demonstrate here that retinoic acid induces CRABP-II messenger RNA in human dermal fibroblasts in a dose-dependent manner. Moreover, we show that the induction by RA can be inhibited by actinomycin D, suggesting that the up-regulation may be mediated by a transcriptional mechanism. We further demonstrate that cycloheximide also has an effect on the up-regulation, suggesting a role for protein synthesis in the regulation of CRABP-II gene expression. We show that CRABP-II is a very stable messenger RNA species, in contrast to the mRNAs for RARα, RARβ, and RARγ. Of interest, we demonstrate no significant difference in the expression of CRABP-II between presenescent and senescent fibroblasts. Taken together, these data suggest that retinoic acid plays a central role in the regulation of CRABP-II gene expression in the dermal fibroblast and that this molecule is the major mediator of the cytoplasmic effects of retinoids in dermal fibroblasts. However, in contrast to RARβ2, there is no apparent change in the regulation of CRABP-II in senescent dermal fibroblasts.

The Cellular and Molecular Biology of Skin Aging

The dramatic alterations in the appearance of the integument with increasing age are due in part to a progressive destruction of the delicate architecture of the connective tissue components of the dermis. Both collagenous and elastic components display a degeneration consistent with the overexpression of proteolytic activity. Recent advances in the field of molecular gerontology, using in vitro models of cellular aging, are yielding clues as to the fundamental causes of dermal aging. Dermal fibroblasts possess a finite replicative capacity of 50 to 100 doublings, then cease replicating in response to growth factors. Cells cultivated to the end of their replicative lifespan in vitro display alterations consistent with their playing a role in aging in vivo. In particular, senescent dermal fibroblasts overexpress metalloproteinase activities that may explain the age-related atrophy of extracellular matrix architecture. The recent discovery of a structural change in the telomeric region of the genome with cellular aging and new insights into DNA damage checkpoint mechanisms offer new opportunities to uncover both the molecular mechanisms regulating cellular aging and possibly to devise new strategies to manipulate these molecular events for therapeutic effect.

Ultrastructure of dermal fibroblasts during development and aging: Relationship to in vitro senescence of dermal fibroblasts

One approach to understanding the relationship between in vitro cellular senescence and in vivo aging is to define the development and aging of cells in vivo and then to compare these in vivo properties with the in vitro behavior of the same cells. The Syrian hamster is being used as an experimental aging model to investigate the intrinsic developmental program of dermal fibroblasts in vivo (proliferation, extracellular matrix (ECM) production, quiescence, and reactivation during wound repair) in order to determine whether the in vivo differentiation program and mature function of these cells is related to their in vitro proliferation and senescence pattern. The ultrastructure of dermal fibroblasts from midfetal development through old age is described, and a working hypothesis of the development and aging of dermal fibroblasts is proposed as a framework for further evaluation of the relationship between in vitro proliferative capacity of dermal fibroblasts and in vivo developmental and age-related changes in the dermis.

Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts

A longitudinal study was performed to confirm the inverse relationship between in vivo age and in vitro proliferative capacity previously observed in a cross-sectional study, and to investigate the relationship between the growth of dermal fibroblasts in vitro and a physiological function (i.e., wound repair) that is known to decline with age in vivo. Fibroblast cultures were generated from skin punch biopsies from 12 male hamsters beginning at 1 month of age and at 6-months interval thereafter until the natural death of the animal. All cultures from all individuals exhibited finite proliferative capacity, and an inverse relationship was observed between donor age and maximum in vitro proliferative capacity. In addition, a direct correlation between the in vitro proliferative capacity of the dermal fibroblasts in vitro and the repair efficiency of the biopsy site was observed. However, these changes in the in vitro proliferative capacity and in vivo wound repair efficiency were not progressive beyond 12–18 months of age and were not indicative at any age of an individual's ultimate lifespan. This study provides evidence that in vitro proliferative capacity of dermal fibroblasts and in vivo wound repair may be comparable phenomena that share a common mechanism. However, the nonprogressive nature and the lack of correlation between these phenomena and the individual's ultimate lifespan indicate that their use as biological markers of aging is limited to animals younger than the mean lifespan of the species.