Senescent Human Fibroblasts Research Articles

517 Regulation of the lipidome and lipid metabolism in prematurely senescent human dermal fibroblasts

A wide range of environmental factors (e.g. UV radiation, pollution) challenge skin homeostasis and it’s prolonged imbalance accelerates skin aging. Aged skin is associated with accumulation of senescent cells which harbor damaged biomolecules and expose danger signals and inflammatory mediators. Prolonged tissue presence of senescent cells negatively affects their microenvironment and contributes to age related tissue decline. Global lipidomics with an UHPLC triple quadrupole MS/MS method confirmed previously reported increase of lysophospholipids in stress-induced premature senescent (SIPS) fibroblasts in vitro and additionally revealed that phospholipids are not the only senescence regulated lipids.

A focused natural compound screen reveals senolytic and senostatic effects of Isatis tinctoria

ABSTRACT Natural products and their derivatives historically represent alternatives to conventional synthetic molecules for pharmacotherapy, ranging from cancer chemotherapeutics to cosmetic ingredients that exert anti-aging activities. Cellular senescence is considered a main driver of skin aging, yet natural products that target skin senescence in a specific manner are not thoroughly explored. Here, we performed a focused compound screen to identify natural products that exert anti-senescence effects. We found that Isatis tinctoria, woad extracts, displayed a senolytic effect on senescent human skin fibroblasts. Furthermore, treatment with woad extracts attenuated the expression of pro-inflammatory senescence-associated secretory phenotype (SASP), showing a senostatic activity. Intriguingly, woad extracts displayed only a marginal cytotoxic effect toward senescent human lung fibroblasts. Thus, our results reveal the potential activities of woad extracts for targeting skin senescence and suggest that woad extracts could be an attractive ingredient for cosmetics to prevent skin aging.

Anti-Aging Effect and Mechanism of Proanthocyanidins Extracted from Sea buckthorn on Hydrogen Peroxide-Induced Aging Human Skin Fibroblasts.

Oxidative stress is the leading cause of skin aging damage. Excessive accumulation of reactive oxygen species (ROS) in cells induced by hydrogen peroxide (H2O2) triggers a decrease in collagen synthesis and an increase in collagen degradation, which are biomarkers of skin aging. We evaluated the potential protective mechanism of Sea buckthorn proanthocyanidins (SBP) against the oxidative stress-induced skin aging process from multiple aspects. We treated human skin fibroblasts (HSFs) with 300 µmoL/L of H2O2 for 24 h, followed by 25, 50, and 100 µg/mL of SBP for 24 h. The results showed that SBP could enhance the activities of superoxide dismutase (SOD) and glutathione (GSH), effectively remove excess ROS, and significantly improve the changes in cell morphology and viability caused by excessive ROS in skin cells. In addition, SBP could promote the synthesis of Col I in aging HSFs through the TGF-β1/Smads pathway and inhibit the degradation of Col I by regulating the MMPs/TIMPs system, thereby maintaining the stability of the ECM structure to achieve anti-aging purposes. Finally, we studied the migration ability of SBP, and the results showed that 100 µg/mL of SBP was most conducive to the cell migration of senescent cells, laying a foundation for follow-up animal experiments. These results will increase the application value of SBP in the cosmetic and antioxidative functional food industries.

Activation of Nrf2 in fibroblasts promotes a skin aging phenotype via an Nrf2-miRNA-collagen axis

Aging is associated with progressive skin fragility and a tendency to tear, which can lead to severe clinical complications. The transcription factor NRF2 is a key regulator of the cellular antioxidant response, and pharmacological NRF2 activation is a promising strategy for the prevention of age-related diseases. Using a combination of molecular and cellular biology, histology, imaging and biomechanical studies we show, however, that constitutive genetic activation of Nrf2 in fibroblasts of mice suppresses collagen and elastin expression, resulting in reduced skin strength as seen in aged mice. Mechanistically, the “aging matrisome” results in part from direct Nrf2-mediated overexpression of a network of microRNAs that target mRNAs of major skin collagens and other matrix components. Bioinformatics and functional studies revealed high NRF2 activity in aged human fibroblasts in 3D skin equivalents and human skin biopsies, highlighting the translational relevance of the functional mouse data. Together, these results identify activated NRF2 as a promoter of age-related molecular and biomechanical skin features.

Cathepsin F is a potential marker for senescent human skin fibroblasts and keratinocytes associated with skin aging

Cellular senescence is characterized by cell cycle arrest and the senescence-associated secretory phenotype (SASP) and can be triggered by a variety of stimuli, including deoxyribonucleic acid (DNA) damage, oxidative stress, and telomere exhaustion. Cellular senescence is associated with skin aging, and identification of specific markers of senescent cells is essential for development of targeted therapies. Cathepsin F (CTSF) has been implicated in dermatitis and various cancers and participates in cell immortalization through its association with Bcl family proteins. It is a candidate therapeutic target to specifically label and eliminate human skin fibroblasts and keratinocytes immortalized by aging and achieve skin rejuvenation. In this study, we investigated whether CTSF is associated with senescence in human fibroblasts and keratinocytes. In senescence models, created using replicative aging, ionizing radiation exposure, and the anticancer drug doxorubicin, various senescence markers were observed, such as senescence-associated β-galactosidase (SA-β-gal) activity, increased SASP gene expression, and decreased uptake of the proliferation marker BrdU. Furthermore, CTSF expression was elevated at the gene and protein levels. In addition, CTSF-positive cells were abundant in aged human epidermis and in some parts of the dermis. In the population of senescent cells with arrested division, the number of CTSF-positive cells was significantly higher than that in the proliferating cell population. These results suggest that CTSF is a candidate for therapeutic modalities targeting aging fibroblasts and keratinocytes.

A conserved long-distance telomeric silencing mechanism suppresses mTOR signaling in aging human fibroblasts.

Telomeres are repetitive nucleotide sequences at the ends of each chromosome. It has been hypothesized that telomere attrition evolved as a tumor suppressor mechanism in large long-lived species. Long telomeres can silence genes millions of bases away through a looping mechanism called telomere position effect over long distances (TPE-OLD). The function of this silencing mechanism is unknown. We determined a set of 2322 genes with high positional conservation across replicatively aging species that includes known and candidate TPE-OLD genes that may mitigate potentially harmful effects of replicative aging. Notably, we identified PPP2R2C as a tumor suppressor gene, whose up-regulation by TPE-OLD in aged human fibroblasts leads to dephosphorylation of p70S6 kinase and mammalian target of rapamycin suppression. A mechanistic link between telomeres and a tumor suppressor mechanism supports the hypothesis that replicative aging fulfills a tumor suppressor function and motivates previously unknown antitumor and antiaging strategies.

Temporal changes in mitochondrial function and reactive oxygen species generation during the development of replicative senescence in human fibroblasts.

Mitochondria are dysfunctional in post-senescent cells. Therefore, age-dependent impairment of mitochondrial energy production accompanied by excessive mitochondrial reactive oxygen species (ROS) is proposed to be a key driver of cellular senescence, which is a state of irreversible cell cycle arrest. However, it remains to be clarified whether mitochondrial dysfunction initiates or accelerates replicative senescence. In this study, we observed no increase in mitochondrial ROS or decrease in mitochondrial respiratory function in human TIG-1 fibroblasts in the transition phase, during which the population doubling rate gradually decreases due to the development of replicative senescence. The integrated stress response and expression of growth differentiation factor 15, which are triggered by respiratory chain deficiency, were also not induced in the transition phase. Mitochondria were elongated without aberrant cristae structures in the transition phase. Mitophagy-related protein levels started to decrease in the transition phase, but autophagic flux slightly increased during replicative senescence. These results suggest that mitochondrial dysfunction and excessive mitochondrial ROS generation do not occur predominately in the transition phase and may not play a role in the development of replicative senescence in normal diploid TIG-1 fibroblasts.

487 Lipidomic and proteomic analyses identify increased degradation of sphingolipids in prematurely senescent human dermal fibroblasts

A wide range of environmental factors (e.g. UV radiation, pollution) challenges skin homeostasis and its prolonged imbalance accelerates skin aging. Aged skin is characterized by presence of senescent cells, which have a changed appearance and phenotype, accumulate damaged biomolecules and simultaneously produce a mixture of danger signaling molecules which negatively affect their microenvironment and activate the immune response. We previously described increased levels of lysophosphatidylcholines, in both replicative senescent and in in vitro stress-induced premature senescent (SIPS) fibroblasts.

614 Intrinsic versus extrinsic skin aging: Extrinsically differ from intrinsically aged human skin fibroblasts in their metabolic adaptive responses and by carrying a signature of catastrophic failure

Aging of human skin results from genetic (= intrinsic) factors, which affect all parts of human skin, and non-genetic (= extrinsic) factors such as solar radiation, which impact only selected skin areas. For both, age-dependent changes in dermal fibroblasts are relevant. To better understand the relationship between intrinsic and extrinsic skin aging, we analyzed dermal skin fibroblasts which were obtained from intrinsically (NHDFINT) versus extrinsically (NHDFEXT) aged human skin of the same donors of one of three age groups (young, middle-aged and old).

Role of H4K16 acetylation in 53BP1 recruitment to double-strand break sites in in vitro aged cells

Increased frequency of DNA double strand breaks (DSBs) with aging suggests an age-associated decline in DSB repair efficiency, which is also influenced by the epigenetic landscape. H4 acetylation at lysine 16 (H4K16Ac) has been related to DSB repair since deacetylation of this mark is required for efficient 53BP1 recruitment to DSBs. Although age-associated changes in H4K16Ac levels have been studied, their contribution to age-related DSB accumulation remains unknown. In vitro aged Human Dermal Fibroblasts (HDFs) display lower levels of H4K16A that correlate with reduced recruitment of 53BP1 to basal DSBs. Following DNA damage induction, early passage (EP) cells suffered from a transient H4K16 deacetylation that allowed proper 53BP1 recruitment to DSBs. In contrast, to reach this specific and optimum level, aged cells responded by increasing their overall lower H4K16Ac levels. Induced hyperacetylation of late passage (LP) cells using trichostatin A increased H4K16Ac levels but did not ameliorate 53BP1 recruitment. Instead, deacetylation induced by MOF silencing reduced H4K16Ac levels and compromised 53BP1 recruitment in both EP and LP cells. Age-associated decrease of H4K16Ac levels contributes to the repair defect displayed by in vitro aged cells. H4K16Ac responds to DNA damage in order to reach a specific, optimum level that allows proper 53BP1 recruitment. This response may be compromised with age, as LP cells depart from lower H4K16Ac levels. Variations in H4K16Ac following the activation of the DNA damage response and aging point at this histone mark as a key mediator between DNA repair and age-associated chromatin alterations.

Comprehensive investigations of key mitochondrial metabolic changes in senescent human fibroblasts.

There is a paucity of detailed data related to the effect of senescence on the mitochondrial antioxidant capacity and redox state of senescent human cells. Activities of TCA cycle enzymes, respiratory chain complexes, hydrogen peroxide (H2O2), superoxide anions (SA), lipid peroxides (LPO), protein carbonyl content (PCC), thioredoxin reductase 2 (TrxR2), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), reduced glutathione (GSH), and oxidized glutathione (GSSG), along with levels of nicotinamide cofactors and ATP content were measured in young and senescent human foreskin fibroblasts. Primary and senescent cultures were biochemically identified by monitoring the augmented cellular activities of key glycolytic enzymes including phosphofructokinase, lactate dehydrogenase, and glycogen phosphorylase, and accumulation of H2O2, SA, LPO, PCC, and GSSG. Citrate synthase, aconitase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and complex I-III, II-III, and IV activities were significantly diminished in P25 and P35 cells compared to P5 cells. This was accompanied by significant accumulation of mitochondrial H2O2, SA, LPO, and PCC, along with increased transcriptional and enzymatic activities of TrxR2, SOD2, GPx1, and GR. Notably, the GSH/GSSG ratio was significantly reduced whereas NAD+/NADH and NADP+/NADPH ratios were significantly elevated. Metabolic exhaustion was also evident in senescent cells underscored by the severely diminished ATP/ADP ratio. Profound oxidative stress may contribute, at least in part, to senescence pointing at a potential protective role of antioxidants in aging-associated disease.

Senescent cells limit p53 activity via multiple mechanisms to remain viable

Super-enhancers regulate genes with important functions in processes that are cell type-specific or define cell identity. Mouse embryonic fibroblasts establish 40 senescence-associated super-enhancers regardless of how they become senescent, with 50 activated genes located in the vicinity of these enhancers. Here we show, through gene knockdown and analysis of three core biological properties of senescent cells that a relatively large number of senescence-associated super-enhancer-regulated genes promote survival of senescent mouse embryonic fibroblasts. Of these, Mdm2, Rnase4, and Ang act by suppressing p53-mediated apoptosis through various mechanisms that are also engaged in response to DNA damage. MDM2 and RNASE4 transcription is also elevated in human senescent fibroblasts to restrain p53 and promote survival. These insights identify key survival mechanisms of senescent cells and provide molecular entry points for the development of targeted therapeutics that eliminate senescent cells at sites of pathology.

Effects of simulated microgravity on senescent human fibroblasts

This study aimed to assess the effects of simulated microgravity (SMG) on senescent human fibroblasts (HFs). The HFs were treated with 150 µM H2O2 in a culture medium for 90 minutes for senescent induction. The senescent HFs were induced SMG for 72 hours by a Gravity simulator, while the control group was treated in the same CO2 incubator. Cell morphology analysis showed that the SMG condition retrieved the fibroblastic morphology from the senescence. WST1 assay and cell counting indicated that the senescent HFs viability from the SMG group was higher than the control group (OD value of 0.80 ± 0.13 vs. 0.64 ± 0.11, respectively). In cell cycle progression, the G0/G1 cell percentage of senescent HFs from the SMG group was reduced, compared to the control group (45.84 ± 1.37% vs. 57.22 ± 1.56%, respectively). The senescent HFs from the SMG group exhibited a lower nuclear area than the control group (186 ± 4 µm2 vs. 217 ± 8 µm2, respectively). This resulted in the performance of a higher nuclear shape value of senescent HFs from SMG, compared to the control group (0.74 ± 0.02 vs. 0.70 ± 0.01, respectively). SMG condition caused the down-regulation of the senescence-related transcript. These results found that SMG could regain the normal characteristics of HFs from the senescence.

Abstract 3638: Age-related changes in pancreatic fibroblasts promote growth and progression of pancreatic cancer

Abstract Aging is an important independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the ways in which aging alters cell populations that comprise the PDAC tumor microenvironment (TME) and impacts tumor growth are incompletely understood. PDAC tumors are highly desmoplastic, and fibroblasts are a key component of the TME that are known to alter the behavior of cancer cells and impact treatment responses. Here, we report that normal pancreatic fibroblasts acquire tumor-promoting properties during aging and alter the properties of PDAC cells both in vitro and in vivo. To determine if PDAC tumors may be influenced by an aged microenvironment, we performed orthotopic KPC cell injections into the pancreata of aged (>64-week-old) and young (6-8-week-old) syngeneic C57Bl/6J mice. Aged mice have significantly larger tumors, increased incidence of metastases, and decreased survival compared to young mice. Tumors from aged mice also have increased neovascularization as evidenced by increased CD31 and CD105 expression. We next queried whether aged pancreatic fibroblasts may contribute to this increase in tumor growth. We have generated a panel of aged (donors >55 years old) and young (donors <35 years old) normal human pancreatic fibroblasts to determine the effects of healthy aging fibroblasts on PDAC cell behavior. Conditioned media from aged human pancreatic fibroblasts significantly increases the proliferation of PDAC cells suggesting that secreted factors produced by aged fibroblasts enhance cancer cell growth. We confirmed this finding using the KPC mouse cell line and conditioned media from aged (>68-week-old) or young (6-8-week-old) normal mouse pancreatic fibroblasts, providing evidence that aged fibroblasts may contribute to the increased tumor growth observed in our in vivo experiments. In addition, aged fibroblast conditioned media increases the migratory potential of PDAC cells in vitro. Next, we evaluated changes in the invasiveness of PDAC cells in a 3D co-culture system with either aged or young pancreatic fibroblasts. PDAC spheroids containing cancer cells and aged fibroblasts exhibit a significant increase in invasiveness compared to those containing young fibroblasts. In conclusion, we show that aged, non-cancer-associated pancreatic fibroblasts have the potential to promote growth, migration, and invasiveness in multiple models of pancreatic ductal adenocarcinoma. We hypothesize that factors secreted by pancreatic fibroblast change during aging and that this aged secretome is tumor-promoting. We will present these and ongoing studies examining age-related changes in pancreatic fibroblasts in the context of PDAC tumorigenesis. Citation Format: Daniel J. Zabransky, Yash Chhabra, Mitchell E. Fane, Daniel Delitto, Jacquelyn W. Zimmermann, Elizabeth M. Jaffee, Ashani T. Weeraratna. Age-related changes in pancreatic fibroblasts promote growth and progression of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3638.

Selective Elimination of Senescent Fibroblasts by Targeting the Cell Surface Protein ACKR3.

The accumulation of senescent cells in aging tissues is associated with age-related diseases and functional decline. Thus, senolysis, a therapy aimed at rejuvenation by removing senescent cells from the body, is being developed. However, this therapy requires the identification of membrane surface antigens that are specifically expressed on senescent cells for their selective elimination. We showed that atypical chemokine receptor 3 (ACKR3), a receptor of the CXC motif chemokine 12 (CXCL12) implicated in cancer, inflammation, and cardiovascular disorders, is selectively expressed on the surface of senescent human fibroblasts but not on proliferating cells. Importantly, the differential presence of ACKR3 enabled the isolation of senescent cells by flow cytometry using anti-ACKR3 antibodies. Furthermore, antibody-dependent cellular cytotoxicity assays revealed that cell surface ACKR3 preferentially sensitizes senescent but not dividing fibroblasts to cell injury by natural killer cells. Conclusively, the selective expression of ACKR3 on the surface of senescent cells allows the preferential elimination of senescent cells. These results might contribute to the future development of novel senolysis approaches.

Shotgun proteomics of extracellular matrix in late senescent human dermal fibroblasts reveals a down-regulated fibronectin-centered network

Extracellular matrix (ECM) proteins play a pivotal role in cell growth and differentiation. To characterize aged ECM proteins, we compared the proteomes by shotgun method of young (passage #15) and late senescent (passage #40) human dermal fibroblasts (HDFs) using SDS-PAGE coupled with LC–MS/MS. The relative abundance of identified proteins was determined using mol% of individual proteins as a semi-quantitative index. Fifteen ECM proteins including apolipoprotein B (APOB) and high-temperature requirement factor 1 (HTRA1) were up-regulated, whereas 50 proteins including fibronectin 1 (FN1) and vitronectin (VTN) were down-regulated in late senescent HDFs. The identified ECM proteins combined with plasma membrane were queried to construct the protein–protein interaction network using Ingenuity Pathways Analysis, resulting in a distinct FN1-centered network. Of differentially abundant ECM proteins in shotgun proteomics, the protein levels of FN1, VTN, APOB, and HTRA1 were verified by immunoblot analysis. The results suggest that the aging process in HDFs might be finally involved in the impaired FN1 regulatory ECM network combined with altered interaction of neighboring proteins. Shotgun proteomics of highly aged HDFs provides insight for further studies of late senescence-related alterations in ECM proteins.

Inhibition of matrix metalloproteinase expression by selective clearing of senescent dermal fibroblasts attenuates ultraviolet-induced photoaging.

Photoaging mainly occurs due to ultraviolet (UV) radiation, and is accompanied by increased secretion of matrix metalloproteinases (MMPs) and degradation of collagen. UV radiation induces cell senescence in the skin; however, the role of senescent cells in photoaging remains unclear. Therefore, to elucidate the role of senescent cells in photoaging, we evaluated the effect of senolytics in a photoaging mouse model and investigated the underlying mechanism of their antiaging effect. Both UV-induced senescent human dermal fibroblasts and a photoaging mouse model, ABT-263 and ABT-737, demonstrated senolytic effects on senescent fibroblasts. Moreover, we found that several senescence-associated secretory phenotype factors, such as IL-6, CCL5, CCL7, CXCL12, and SCF, induced MMP-1 expression in dermal fibroblasts, which decreased after treatment with ABT-263 and ABT-737 in vivo and in vitro. Both senolytic drugs attenuated the induction of MMPs and decreased collagen density in the photoaging mouse model. Our data suggest that senolytic agents reduce UV-induced photoaging, making strategies for targeting senescent dermal fibroblasts promising options for the treatment of photoaging.

Pentoxifylline Inhibits Pulmonary Fibrosis by Regulating Cellular Senescence in Mice.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive disease, and its occurrence and development are mediated by cellular senescence. Drugs targeting senescent cells seem like a promising and efficacious strategy for IPF treatment. Previous studies have illustrated that pentoxifylline (PTX) may play a certain role in inhibiting pulmonary fibrosis and combating cellular senescence. In this study, we demonstrated that PTX administration inhibits pulmonary fibrosis development and cellular senescence in the bleomycin (BLM)-induced IPF mice model. Moreover, the expression levels of fibrosis-related genes and senescence-related genes in mice lung tissue and primary pulmonary fibroblasts illustrated lung fibroblasts’ vital role in these two processes. And the curative effect of PTX was completed mainly by acting on lung fibroblasts. Besides, during the whole treatment, delayed initiation or advanced halt of PTX administration would influence its effectiveness in reducing fibrotic and senescent traits in various degrees, and the latter influenced more. We further determined that a long period of PTX administration could bring noticeable benefits to mice in recovering BLM-induced lung fibrosis and suppressing age-associated cellular senescence. Moreover, it was still effective when PTX administration was used to treat senescent human fibroblasts. Thus, our findings manifested that PTX therapy is an efficient remedy for pulmonary fibrosis by suppressing cellular senescence.

Unveiling a novel serpinB2-tripeptidyl peptidase II signaling axis during senescence.

Tripeptidyl peptidase II (TPPII or TPP2) degrades N-terminal tripeptides from proteins and peptides. Studies in both humans and mice have shown that TPPII deficiency is linked to cellular immune-senescence, lifespan regulation and the aging process. However, the mechanism of how TPPII participates in these processes is less clear. In this study, we established a chemical probe-based assay and found that although the mRNA and protein levels of TPPII were not altered during senescence, its enzymatic activity was reduced in senescent human fibroblasts. We also showed that elevation of the levels of the serine protease inhibitor serpinB2 reduced TPPII activity in senescent cells. Moreover, suppression of TPPII led to elevation in the amount of lysosomal contents as in well as TPPI (TPP1) and β-galactosidase activities, suggesting that lysosome biogenesis is induced to compensate for the reduction of TPPII activity in senescent cells. Together, this study discloses a critical role of the serpinB2-TPPII signaling pathway in proteostasis during senescence. Since serpinB2 levels can be increased by a variety of cellular stresses, reduction of TPPII activity through activation of serpinB2 might represent a common pathway for cells to respond to different stress conditions. This article has an associated First Person interview with the first author of the paper.

Isatis tinctoria L. Leaf Extract Inhibits Replicative Senescence in Dermal Fibroblasts by Regulating mTOR-NF-κB-SASP Signaling.

Senescent fibroblasts progressively deteriorate the functional properties of skin tissue. Senescent cells secrete senescence-associated secretory phenotype (SASP) factor, which causes the aging of surrounding non-senescent cells and accelerates aging in the individuals. Recent findings suggested the senomorphic targeting of the SASP regulation as a new generation of effective therapeutics. We investigated whether Isatis tinctoria L. leaf extract (ITE) inhibited senescence biomarkers p53, p21CDKN1A, and p16INK4A gene expression, and SASP secretions by inhibiting cellular senescence in the replicative senescent human dermal fibroblast (RS-HDF). ITE has been demonstrated to inhibit the secretion of SASP factors in several senomorphic types by regulating the MAPK/NF-κB pathway via its inhibitory effect on mTOR. ITE suppressed the inflammatory response by inhibiting mTOR, MAPK, and IκBα phosphorylation, and blocking the nuclear translocation of NF-κB. In addition, we observed that autophagy pathway was related to inhibitory effect of ITE on cellular senescence. From these results, we concluded that ITE can prevent and restore senescence by blocking the activation and secretion of senescence-related factors generated from RS-HDFs through mTOR-NF-κB regulation.