The role of lipid-based signalling in wound healing and senescence.

Lipid-based signalling modulates several cellular processes and intercellular communication during wound healing and tissue regeneration. Bioactive lipids include but are not limited to the diverse group of eicosanoids, phospholipids, and extracellular vesicles and mediate the attraction of immune cells, initiation of inflammatory responses, and their resolution. In aged individuals, wound healing and tissue regeneration are greatly impaired, resulting in a delayed healing process and non-healing wounds. Senescent cells accumulate with age in vivo, preferably at sites implicated in age-associated pathologies and their elimination was shown to alleviate many age-associated diseases and disorders. In contrast to these findings, the transient presence of senescent cells in the process of wound healing exerts beneficial effects and limits fibrosis. Hence, clearance of senescent cells during wound healing was repeatedly shown to delay wound closure in vivo. Recent findings established a dysregulated synthesis of eicosanoids, phospholipids and extracellular vesicles as part of the senescent phenotype. This intriguing connection between cellular senescence, lipid-based signalling, and the process of wound healing and tissue regeneration prompts us to compile the current knowledge in this review and propose future directions for investigation.

Circulating miR-19a-3p and miR-19b-3p characterize the human aging process and their isomiRs associate with healthy status at extreme ages.

Blood circulating microRNAs (c-miRs) are potential biomarkers to trace aging and longevity trajectories to identify molecular targets for anti-aging therapies. Based on a cross-sectional study, a discovery phase was performed on 12 donors divided into four groups: young, old, healthy, and unhealthy centenarians. The identification of healthy and unhealthy phenotype was based on cognitive performance and capabilities to perform daily activities. Small RNA sequencing identified 79 differentially expressed c-miRs when comparing young, old, healthy centenarians, and unhealthy centenarians. Two miRs, that is, miR-19a-3p and miR-19b-3p, were found increased at old age but decreased at extreme age, as confirmed by RT-qPCR in 49 donors of validation phase. The significant decrease of those miR levels in healthy compared to unhealthy centenarians appears to be due to the presence of isomiRs, not detectable with RT-qPCR, but only with a high-resolution technique such as deep sequencing. Bioinformatically, three main common targets of miR-19a/b-3p were identified, that is, SMAD4, PTEN, and BCL2L11, converging into the FoxO signaling pathway, known to have a significant role in aging mechanisms. For the first time, this study shows the age-related increase of plasma miR-19a/b-3p in old subjects but a decrease in centenarians. This decrease is more pronounced in healthy centenarians and was confirmed by the modified pattern of isomiRs comparing healthy and unhealthy centenarians. Thus, our study paves the way for functional studies using c-miRs and isomiRs as additional parameter to track the onset of aging and age-related diseases using new potential biomarkers.

Epilipidomics of Senescent Dermal Fibroblasts Identify Lysophosphatidylcholines as Pleiotropic Senescence-Associated Secretory Phenotype (SASP) Factors.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome-the oxidative modifications of phospholipids-of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging.

Organotypic human skin culture models constructed with senescent fibroblasts show hallmarks of skin aging.

Skin aging is driven by intrinsic and extrinsic factors impacting on skin functionality with progressive age. One factor of this multifaceted process is cellular senescence, as it has recently been identified to contribute to a declining tissue functionality in old age. In the skin, senescent cells have been found to markedly accumulate with age, and thus might impact directly on skin characteristics. Especially the switch from young, extracellular matrix-building fibroblasts to a senescence-associated secretory phenotype (SASP) could alter the microenvironment in the skin drastically and therefore promote skin aging. In order to study the influence of senescence in human skin, 3D organotypic cultures are a well-suited model system. However, only few "aged" skin- equivalent (SE) models are available, requiring complex and long-term experimental setups. Here, we adapted a previously published full-thickness SE model by seeding increasing ratios of stress-induced premature senescent versus normal fibroblasts into the collagen matrix, terming these SE "senoskin". Immunohistochemistry stainings revealed a shift in the balance between proliferation (Ki67) and differentiation (Keratin 10 and Filaggrin) of keratinocytes within our senoskin equivalents, as well as partial impairment of skin barrier function and changed surface properties. Monitoring of cytokine levels of known SASP factors confirmedly showed an upregulation in 2D cultures of senescent cells and at the time of seeding into the skin equivalent. Surprisingly, we find a blunted response of cytokines in the senoskin equivalent over time during 3D differentiation.

Extracellular Vesicles in Human Skin: Cross-Talk from Senescent Fibroblasts to Keratinocytes by miRNAs.

Extracellular vesicles (EVs) and their miRNA cargo are intercellular communicators transmitting their pleiotropic messages between different cell types, tissues, and body fluids. Recently, they have been reported to contribute to skin homeostasis and were identified as members of the senescence-associated secretory phenotype of human dermal fibroblasts. However, the role of EV-miRNAs in paracrine signaling during skin aging is yet unclear. Here we provide evidence for the existence of small EVs in the human skin and dermal interstitial fluid using dermal open flow microperfusion and show that EVs and miRNAs are transferred from dermal fibroblasts to epidermal keratinocytes in 2D cell culture and in human skin equivalents. We further show that the transient presence of senescent fibroblast derived small EVs accelerates scratch closure of epidermal keratinocytes, whereas long-term incubation impairs keratinocyte differentiation in vitro. Finally, we identify vesicular miR-23a-3p, highly secreted by senescent fibroblasts, as one contributor of the EV-mediated effect on keratinocytes in in vitro wound healing assays. To summarize, our findings support the current view that EVs and their miRNA cargo are members of the senescence-associated secretory phenotype and, thus, regulators of human skin homeostasis during aging.

Small extracellular vesicles and their miRNA cargo are anti-apoptotic members of the senescence-associated secretory phenotype.

Loss of functionality during aging of cells and organisms is caused and accompanied by altered cell-to-cell communication and signalling. One factor thereby is the chronic accumulation of senescent cells and the concomitant senescence-associated secretory phenotype (SASP) that contributes to microenvironment remodelling and a pro-inflammatory status. While protein based SASP factors have been well characterized, little is known about small extracellular vesicles (sEVs) and their miRNA cargo. Therefore, we analysed secretion of sEVs from senescent human dermal fibroblasts and catalogued the therein contained miRNAs. We observed a four-fold increase of sEVs, with a concomitant increase of >80% of all cargo miRNAs. The most abundantly secreted miRNAs were predicted to collectively target mRNAs of pro-apoptotic proteins, and indeed, senescent cell derived sEVs exerted anti-apoptotic activity. In addition, we identified senescence-specific differences in miRNA composition of sEVs, with an increase of miR-23a-5p and miR-137 and a decrease of miR-625-3p, miR-766-3p, miR-199b-5p, miR-381-3p, miR-17-3p. By correlating intracellular and sEV-miRNAs, we identified miRNAs selectively retained in senescent cells (miR-21-3p and miR-17-3p) or packaged specifically into senescent cell derived sEVs (miR-15b-5p and miR-30a-3p). Therefore, we suggest sEVs and their miRNA cargo to be novel, members of the SASP that are selectively secreted or retained in cellular senescence.

Blocking negative effects of senescence in human skin fibroblasts with a plant extract.

There is increasing evidence that senescent cells are a driving force behind many age-related pathologies and that their selective elimination increases the life- and healthspan of mice. Senescent cells negatively affect their surrounding tissue by losing their cell specific functionality and by secreting a pro-tumorigenic and pro-inflammatory mixture of growth hormones, chemokines, cytokines and proteases, termed the senescence-associated secretory phenotype (SASP). Here we identified an extract from the plant Solidago virgaurea subsp. alpestris, which exhibited weak senolytic activity, delayed the acquisition of a senescent phenotype and induced a papillary phenotype with improved functionality in human dermal fibroblasts. When administered to stress-induced premature senescent fibroblasts, this extract changed their global mRNA expression profile and particularly reduced the expression of various SASP components, thereby ameliorating the negative influence on nearby cells. Thus, the investigated plant extract represents a promising possibility to block age-related loss of tissue functionality.

Autophagy deficient keratinocytes display increased DNA damage, senescence and aberrant lipid composition after oxidative stress in vitro and in vivo.

Autophagy allows cells fundamental adaptations to metabolic needs and to stress. Using autophagic bulk degradation cells can clear crosslinked macromolecules and damaged organelles that arise under redox stress. Accumulation of such debris results in cellular dysfunction and is observed in aged tissue and senescent cells. Conversely, promising anti-aging strategies aim at inhibiting the mTOR pathway and thereby activating autophagy, to counteract aging associated damage. We have inactivated autophagy related 7 (Atg7), an essential autophagy gene, in murine keratinocytes (KC) and have found in an earlier study that this resulted in increased baseline oxidative stress and reduced capacity to degrade crosslinked proteins after oxidative ultraviolet stress. To investigate whether autophagy deficiency would promote cellular aging, we studied how Atg7 deficient (KO) and Atg7 bearing cells (WT) would respond to stress induced by paraquat (PQ), an oxidant drug commonly used to induce cellular senescence. Atg7 deficient KC displayed increased prostanoid signaling and a pro- mitotic gene expression signature as compared to the WT. After exposure to PQ, both WT and KO cells showed an inflammatory and stress-related transcriptomic response. However, the Atg7 deficient cells additionally showed drastic DNA damage- and cell cycle arrest signaling. Indeed, DNA fragmentation and -oxidation were strongly increased in the stressed Atg7 deficient cells upon PQ stress but also after oxidizing ultraviolet A irradiation. Damage associated phosphorylated histone H2AX (γH2AX) foci were increased in the nuclei, whereas expression of the nuclear lamina protein lamin B1 was strongly decreased. Similarly, in both, PQ treated mouse tail skin explants and in UVA irradiated mouse tail skin, we found a strong increase in γH2AX positive nuclei within the basal layer of Atg7 deficient epidermis. Atg7 deficiency significantly affected expression of lipid metabolic genes. Therefore we performed lipid profiling of keratinocytes which demonstrated a major dysregulation of cellular lipid metabolism. We found accumulation of autophagy agonisitic free fatty acids, whereas triglyceride levels were strongly decreased. Together, our data show that in absence of Atg7/autophagy the resistance of keratinocytes to intrinsic and environmental oxidative stress was severely impaired and resulted in DNA damage, cell cycle arrest and a disturbed lipid phenotype, all typical for premature cell aging.

SNEVhPrp19/hPso4 Regulates Adipogenesis of Human Adipose Stromal Cells.

Aging is accompanied by loss of subcutaneous adipose tissue. This may be due to reduced differentiation capacity or deficiency in DNA damage repair (DDR) factors. Here we investigated the role of SNEVhPrp19/hPso4, which was implicated in DDR and senescence evasion, in adipogenic differentiation of human adipose stromal cells (hASCs). We showed that SNEV is induced during adipogenesis and localized both in the nucleus and in the cytoplasm. Knockdown of SNEV perturbed adipogenic differentiation and led to accumulation of DNA damage in hASCs upon oxidative stress. In addition, we demonstrated that SNEV is required for fat deposition in Caenorhabditis elegans. Consequently, we tested other DDR factors and found that WRN is also required for adipogenesis in both models. These results demonstrate that SNEV regulates adipogenesis in hASCs and indicate that DDR capacity in general might be a pre-requisite for this process.

High levels of oncomiR-21 contribute to the senescence-induced growth arrest in normal human cells and its knock-down increases the replicative lifespan.

Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21(CIP1) and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21(CIP) (1) , and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.