Senescence-associated alterations in the extracellular matrix: deciphering their role in the regulation of cellular function.

The extracellular matrix (ECM) is a dynamic structural network that provides a physical scaffolding, as well as biochemical factors that maintain normal tissue homeostasis and thus its disruption is implicated in many pathological conditions. On the other hand, senescent cells express a particular secretory phenotype, affecting the composition and organization of the surrounding ECM and modulating their microenvironment. As accumulation of senescent cells may be linked to the manifestation of several age-related conditions, senescence-associated ECM alterations may serve as targets for novel anti-aging treatment modalities. Here, we will review characteristic changes in the ECM elicited by cellular senescence and we will discuss the complex interplay between ECM and senescent cells, in relation to normal aging and selected age-associated pathologies.

Decreased differentiation capacity and altered expression of extracellular matrix components in irradiation-mediated senescent human breast adipose-derived stem cells.

Radiotherapy is widely used for the treatment of breast cancer. However, we have shown that ionizing radiation can provoke premature senescence in breast stromal cells. In particular, breast stromal fibroblasts can become senescent after irradiation both in vitro and in vivo and they express an inflammatory phenotype and an altered profile of extracellular matrix components, thus facilitating tumor progression. Adipose-derived stem cells (ASCs) represent another major component of the breast tissue stroma. They are multipotent cells and due to their ability to differentiate in multiple cell lineages they play an important role in tissue maintenance and repair in normal and pathologic conditions. Here, we investigated the characteristics of human breast ASCs that became senescent prematurely after their exposure to ionizing radiation. We found decreased expression levels of the specific mesenchymal cell surface markers CD105, CD73, CD44, and CD90. In parallel, we demonstrated a significantly reduced expression of transcription factors regulating osteogenic (i.e., RUNX2), adipogenic (i.e., PPARγ), and chondrogenic (i.e., SOX9) differentiation; this was followed by an analogous reduction in their differentiation capacity. Furthermore, they overexpress inflammatory markers, that is, IL-6, IL-8, and ICAM-1, and a catabolic phenotype, marked by the reduction of collagen type I and the increase of MMP-1 and MMP-13 expression. Finally, we detected changes in proteoglycan expression, for example, the upregulation of syndecan 1 and syndecan 4 and the downregulation of decorin. Notably, all these alterations, when observed in the breast stroma, represent poor prognostic factors for tumor development. In conclusion, we showed that ionizing radiation-mediated prematurely senescent human breast ASCs have a decreased differentiation potential and express specific changes adding to the formation of a permissive environment for tumor growth.

Reacquisition of a spindle cell shape does not lead to the restoration of a youthful state in senescent human skin fibroblasts.

Senescent fibroblasts are characterized by their inability to proliferate and by a pro-inflammatory and catabolic secretory phenotype, which contributes to age-related pathologies. Furthermore, senescent fibroblasts when cultured under classical conditions in vitro are also characterized by striking morphological changes, i.e. they lose the youthful spindle-like appearance and become enlarged and flattened, while their nuclei from elliptical become oversized and highly lobulated. Knowing the strong relation between cell shape and function, we cultured human senescent fibroblasts on photolithographed Si/poly(vinyl alcohol) (PVA) micro-patterned surfaces in order to restore the classical spindle-like geometry and subsequently to investigate whether the changes in senescent cells' morphology are the cause of their functional alterations. Interestingly, under these conditions senescent cells' nuclei do not revert to the classical elliptical phenotype. Furthermore, enforced spindle-shaped senescent cells retained their deteriorated proliferative ability, and maintained the increased gene expression of the cell cycle inhibitors p16Ink4a and p21Waf1. In addition, Si/PVA-patterned-grown senescent fibroblasts preserved their senescence-associated phenotype, as evidenced by the overexpression of inflammatory and catabolic genes such as IL6, IL8, ICAM1 and MMP1 and MMP9 respectively, which was further manifested by an intense downregulation of fibroblasts' most abundant extracellular matrix component Col1A, compared to their young counterparts. These data indicate that the restoration of the spindle-like shape in senescent human fibroblasts is not able to directly alter major functional traits and restore the youthful phenotype.

Short- and long-term treatment with TNF-α inhibits the induction of osteoblastic differentiation in cyclic tensile-stretched periodontal ligament fibroblasts.

BACKGROUND: Cyclic tensile stretching (CTS) induces osteoblastic differentiation of periodontal ligament fibroblasts (PDLF). On the other hand, increased concentrations of tumour necrosis factor-α (TNF-α) are found in inflammatory conditions, leading to periodontal disease and tooth loss. Accordingly, our aim was to investigate the short- and long-term effect of TNF-α on the response of human PDLF to CTS and its implication on osteoblastic differentiation. METHODS: PDLF were either pre-incubated for 4 hours or were repeatedly exposed to TNF-α for up to 50 days and then subjected to CTS. Gene expression was determined by quantitative real-time polymerase chain reaction. Activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and cell proliferation by bromodeoxyuridine incorporation. Intracellular reactive oxygen species were determined by the 2´, 7´-dichlorofluorescein-diacetate assay and osteoblastic differentiation by Alizarin Red-S staining after an osteo-inductive period of 21 days. RESULTS: CTS of PDLF induced an immediate upregulation of the c-fos transcription factor and, further downstream the overexpression of alkaline phosphatase and osteopontin, two major osteoblast marker genes. A 4-hour pre-incubation with TNF-α repressed these effects. Similarly, long-term propagation of PDLF along with TNF-α diminished their osteoblastic differentiation capacity and suppressed cells' CTS-elicited responses. The observed phenomena were not linked with TNF-α-induced premature senescence or oxidative stress. While CTS induced the activation of MAPKs, involved in mechanotransduction, TNF-α treatment provoked a small delay in the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. CONCLUSION: Increased concentrations of TNF-α, such as those recorded in many inflammatory diseases, suppress PDLF's immediate responses to mechanical forces compromising their osteoblastic differentiation potential, possibly leading to tissue's impaired homeostasis.

Effect of hyperglycaemic conditions on the response of human periodontal ligament fibroblasts to mechanical stretching.

OBJECTIVES: The aim of the present study was to investigate the impact of high glucose concentration on the response of human periodontal ligament fibroblasts (PDLFs) to cyclic tensile strain. MATERIALS AND METHODS: Human PDLFs were incubated under normal or high glucose conditions, and then were subjected to cyclic tensile stretching (8 per cent extension, 1 Hz). Gene expression was determined by quantitative real-time polymerase chain reaction. Intracellular reactive oxygen species (ROS) were determined by the 2',7'-dichlorofluorescein-diacetate assay, activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and osteoblastic differentiation was estimated with Alizarin Red-S staining. RESULTS: Cyclic tensile stretching of PDLF leads to an immediate activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), as well as to the increased expression of the transcription factor c-fos, known to regulate many osteogenesis-related genes. At later time points, the alkaline phosphatase and osteopontin genes were also upregulated. Hyperglycaemic conditions inhibited these effects. High glucose conditions were unable to increase ROS levels, but they increased the medium's osmolality. Finally, increase of osmolality mimics the inhibitory effect of hyperglycaemia on MAPK activation, c-fos and osteoblast-specific gene markers' upregulation, as well as osteogenic differentiation capacity. CONCLUSION: Our findings indicate that under high glucose conditions, human PDLFs fail to adequately respond to mechanical deformation, while their strain-elicited osteoblast differentiation ability is deteriorated. The aforementioned effects are most probably mediated by the increased osmolality under hyperglycaemic conditions.

Correction to: Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions.

After publication of the original article, authors found that there has been a minor mistake in the units of kcat and kcat/Km in Table 2. The units should be 103 min-1 g-1 FAE for kcat and mM-1 min-1 g-1 FAE for kcat/Km. This correction does not affect any conclusions drawn within the article.

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions.

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 µM for PFA, 329.9 µM for FA). PFA was not cytotoxic at 0.8-100 µM (IC50 220.23 µM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 µM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

Early responses of human periodontal ligament fibroblasts to cyclic and static mechanical stretching.

Objective: To compare the mechanotransduction caused by cyclic and static mechanical strains in human periodontal ligament fibroblasts (hPDLFs) cultured under identical conditions. Materials and methods: hPDLFs, originating from the same donors, were exposed either to cyclic or to static tensile strain using specially designed devices and under identical culture conditions. Activation of all members of mitogen-activated protein kinases (MAPKs) was monitored by western immunoblot analysis. Expression levels of immediate/early genes c-fos and c-jun were assessed with quantitative real-time polymerase chain reaction. Results: Time course experiments revealed that both types of stresses activate the three members of MAPK, that is ERK, p38, and JNK, with cyclic stress exhibiting a slightly more extended activation. Further downstream, both stresses upregulate the immediate/early genes c-fos and c-jun, encoding components of the activator protein-1 (AP-1), a key transcription factor in osteoblastic differentiation; again cyclic strain provokes a more intense upregulation. Six hours after the application of both strains, MAPK activation and gene expression return to basal levels. Finally, cells exposed to cyclic stress for longer periods are distributed approximately perpendicular to the axis of the applied strain, whereas cells exposed to static loading remain in a random orientation in culture. Conclusion: The findings of the present study indicate similar, although not identical, immediate/early responses of hPDLs to cyclic and static stretching, with cyclic strain provoking a more intense adaptive response of these cells to mechanical deformation.

Design and Synthesis of Novel Antioxidant 2-Substituted-5,7,8-Trimethyl-1,4-Benzoxazine Hybrids: Effects on Young and Senescent Fibroblasts.

The exponential growth of the aged population worldwide is followed by an increase in the prevalence of age-related disorders. Oxidative stress plays central role in damage accumulation during ageing and cell senescence. Thus, a major target of today's anti-ageing research has been focused on antioxidants counteracting senescence. In the current work, six novel 5,7,8-trimethyl-1,4-benzoxazine/catechol or resorcinol hybrids were synthesized connected through a methoxymethyl-1,2,3-triazolyl or a 1,2,3-triazoly linker. The compounds were evaluated for their antioxidant capacity in a cell-free system and for their ability to reduce intracellular ROS levels in human skin fibroblasts, both young (early-passage) and senescent. The most efficient compounds were further tested in these cells for their ability to induce the expression of the gene heme oxygenase-1 (ho-1), known to regulate redox homeostasis, and cellular glutathione (GSH) levels. Overall, the two catechol derivatives were found to be more potent than the resorcinol analogues. Furthermore, these two derivatives were shown to act coordinately as radical scavengers, ROS inhibitors, ho-1 gene expression inducers, and GSH enhancers. Interestingly, one of the two catechol derivatives was also found to enhance human skin fibroblast viability. The properties of the synthesized compounds support their potential use in cosmetic applications, especially in products targeting skin ageing.

Senescent human periodontal ligament fibroblasts after replicative exhaustion or ionizing radiation have a decreased capacity towards osteoblastic differentiation.

Loss of teeth increases with age or after genotoxic treatments, like head and neck radiotherapy, due to periodontium breakdown. Periodontal ligament fibroblasts represent the main cell type in this tissue and are crucial for the maintenance of homeodynamics and for its regeneration. Here, we have studied the characteristics of human periodontal ligament fibroblasts (hPDLF) that became senescent after replicative exhaustion or after exposure to ionizing radiation, as well as their ability for osteoblastic differentiation. We found that senescent hPDLF express classical markers of senescence, as well as a catabolic phenotype, as shown by the decrease in collagen type I and the increase of MMP-2 expression. In addition, we observed a considerably decreased expression of the major transcription factor for osteoblastic differentiation, i.e. Runx2, a down-regulation which was found to be p53-dependent. In accordance to the above, senescent cells have a significantly decreased alkaline phosphatase gene expression and activity, as well as a reduced ability for osteoblastic differentiation, as found by Alizarin Red staining. Interestingly, cells from both type of senescence express similar characteristics, implying analogous functions in vivo. In conclusion, senescent hPDLF express a catabolic phenotype and express a significantly decreased ability towards an osteoblastic differentiation, thus probably affecting tissue development and integrity.

Heterogeneity of Cellular Senescence: Cell Type-Specific and Senescence Stimulus-Dependent Epigenetic Alterations.

The aim of the present study was to provide a comprehensive characterization of whole genome DNA methylation patterns in replicative and ionizing irradiation- or doxorubicin-induced premature senescence, exhaustively exploring epigenetic modifications in three different human cell types: in somatic diploid skin fibroblasts and in bone marrow- and adipose-derived mesenchymal stem cells. With CpG-wise differential analysis, three epigenetic signatures were identified: (a) cell type- and treatment-specific signature; (b) cell type-specific senescence-related signature; and (c) cell type-transversal replicative senescence-related signature. Cluster analysis revealed that only replicative senescent cells created a distinct group reflecting notable alterations in the DNA methylation patterns accompanying this cellular state. Replicative senescence-associated epigenetic changes seemed to be of such an extent that they surpassed interpersonal dissimilarities. Enrichment in pathways linked to the nervous system and involved in the neurological functions was shown after pathway analysis of genes involved in the cell type-transversal replicative senescence-related signature. Although DNA methylation clock analysis provided no statistically significant evidence on epigenetic age acceleration related to senescence, a persistent trend of increased biological age in replicative senescent cultures of all three cell types was observed. Overall, this work indicates the heterogeneity of senescent cells depending on the tissue of origin and the type of senescence inducer that could be putatively translated to a distinct impact on tissue homeostasis.

Down-Regulation of the Proteoglycan Decorin Fills in the Tumor-Promoting Phenotype of Ionizing Radiation-Induced Senescent Human Breast Stromal Fibroblasts.

Down-regulation of the small leucine-rich proteoglycan decorin in the stroma is considered a poor prognostic factor for breast cancer progression. Ionizing radiation, an established treatment for breast cancer, provokes the premature senescence of the adjacent to the tumor stromal fibroblasts. Here, we showed that senescent human breast stromal fibroblasts are characterized by the down-regulation of decorin at the mRNA and protein level, as well as by its decreased deposition in the pericellular extracellular matrix in vitro. Senescence-associated decorin down-regulation is a long-lasting process rather than an immediate response to γ-irradiation. Growth factors were demonstrated to participate in an autocrine manner in decorin down-regulation, with bFGF and VEGF being the critical mediators of the phenomenon. Autophagy inhibition by chloroquine reduced decorin mRNA levels, while autophagy activation using the mTOR inhibitor rapamycin enhanced decorin transcription. Interestingly, the secretome from a series of both untreated and irradiated human breast cancer cell lines with different molecular profiles inhibited decorin expression in young and senescent stromal fibroblasts, which was annulled by SU5402, a bFGF and VEGF inhibitor. The novel phenotypic trait of senescent human breast stromal fibroblasts revealed here is added to their already described cancer-promoting role via the formation of a tumor-permissive environment.

Extracellular matrix alterations in senescent cells and their significance in tissue homeostasis.

Normal cells after a defined number of successive divisions or after exposure to genotoxic stresses are becoming senescent, characterized by a permanent growth arrest. In addition, they secrete increased levels of pro-inflammatory and catabolic mediators, collectively termed "senescence-associated secretory phenotype". Furthermore, senescent cells exhibit an altered expression and organization of many extracellular matrix components, leading to specific remodeling of their microenvironment. In this review we present the current knowledge on extracellular matrix alterations associated with cellular senescence and critically discuss certain characteristic examples, highlighting the ambiguous role of senescent cells in the homeostasis of various tissues under both normal and pathologic conditions.

The role of cellular senescence on the cyclic stretching-mediated activation of MAPK and ALP expression and activity in human periodontal ligament fibroblasts.

Mechanical loading is considered to be a major parameter of the periodontal ligament (PDL) remodeling and differentiation. However, the molecular mechanisms that translate these forces to cellular responses are not fully elucidated. Especially, although aging affects PDL homeostasis, the role of cellular senescence on the activation of signaling pathways in periodontal ligament fibroblasts (PDLF) in response to mechanical stimulation has not been studied yet. Here, we present evidence showing that cyclic mechanical stimulation activates ERK, JNK and p38 MAPK in young (early-passage) human PDLF, in a RhoK-dependent manner. This response was found to be independent of the substratum (i.e. fibronectin or collagen) on which these cells grow. Stretching up-regulates also c-fos, a classical cellular response to mechanical deformation. Inhibition of ERK and JNK reduces, while that of p38 enhances stress-mediated c-fos expression. In addition, cyclic stretching stimulates the expression and activity of alkaline phosphatase (ALP), an early marker of osteoblastic differentiation. We have recently shown that senescent human PDLF have a significantly decreased expression of ALP, linked to an inability towards osteoblastic differentiation. Here, we found that senescent PDLF are able to respond to cyclic mechanical stretching by activating ERK, JNK and p38 MAPK, with similar kinetics compared to young cells, and by up-regulating c-fos and ALP expression and activity. However, even after stimulation, ALP levels in senescent cells are still much lower compared to the basal levels of their young counterparts, suggesting that senescence impairs the differentiation of human PDLF when subjected to cyclic mechanical deformation.

Human lung fibroblasts prematurely senescent after exposure to ionizing radiation enhance the growth of malignant lung epithelial cells in vitro and in vivo.

Cellular senescence, being the result of serial subculturing or of exogenous stresses, is considered to be a potent anticancer mechanism. However, it has been proposed that senescent cells may enhance the growth of adjacent malignant epithelial cells. On the other hand, exposure of tumors to repeated low doses of γ-irradiation is a common treatment regime. Nevertheless, γ-irradiation also affects the neighboring stromal cells and the interaction of the latter with cancer cells. Accordingly, in this study, we have exposed confluent cultures of human lung fibroblasts to repeated subcytotoxic doses of 4 Gy of γ-irradiation. We found that a single dose immediately activates a DNA damage response, leading to an intense, but reversible, cell cycle arrest. After a series of doses (total dose approximately 50 Gy) cellular senescence was accelerated, as shown by permanent growth arrest and the upregulation of specific biochemical and morphological senescence-associated markers. This process was found to be p53-dependent. Next, we studied the effect of these prematurely senescent cells on the growth of human malignant lung cell lines (A549 and H1299) and found that the presence of irradiation-mediated senescent cells strongly enhances the growth of these cancer cells in vitro and in immunocompromised (SCID) mice in vivo. This effect seems not to be related to an induction of epithelial-to-mesenchymal transdifferentiation but, to a significant extent, to the increased expression of matrix metalloproteases (MMPs), as a specific MMP inhibitor significantly restrains the growth of cancer in the presence of senescent fibroblasts. These findings indicate that lung fibroblasts that become senescent after ionizing radiation may contribute to lung cancer progression.