Cellular and extracellular miRNAs are blood‐compartment‐specific diagnostic targets in sepsis

Septic shock is a common medical condition with a mortality approaching 50% where early diagnosis and treatment are of particular importance for patient survival. Novel biomarkers that serve as prompt indicators of sepsis are urgently needed. High‐throughput technologies assessing circulating microRNAs represent an important tool for biomarker identification, but the blood‐compartment specificity of these miRNAs has not yet been investigated. We characterized miRNA profiles from serum exosomes, total serum and blood cells (leukocytes, erythrocytes, platelets) of sepsis patients by next‐generation sequencing and RT‐qPCR (n = 3 × 22) and established differences in miRNA expression between blood compartments. In silico analysis was used to identify compartment‐specific signalling functions of differentially regulated miRNAs in sepsis‐relevant pathways. In septic shock, a total of 77 and 103 miRNAs were down‐ and up‐regulated, respectively. A majority of these regulated miRNAs (14 in serum, 32 in exosomes and 73 in blood cells) had not been previously associated with sepsis. We found a distinctly compartment‐specific regulation of miRNAs between sepsis patients and healthy volunteers. Blood cellular miR‐199b‐5p was identified as a potential early indicator for sepsis and septic shock. miR‐125b‐5p and miR‐26b‐5p were uniquely regulated in exosomes and serum, respectively, while one miRNA (miR‐27b‐3p) was present in all three compartments. The expression of sepsis‐associated miRNAs is compartment‐specific. Exosome‐derived miRNAs contribute significant information regarding sepsis diagnosis and survival prediction and could serve as newly identified targets for the development of novel sepsis biomarkers.     

Exosomal vesicles enhance immunosuppression in chronic inflammation: Impact in cellular senescence and the aging process

Exosomes represent an evolutionarily conserved signaling pathway which can act as an alarming mechanism in responses to diverse stresses, e.g. chronic inflammation activates the budding of exosomal vesicles in both immune and non-immune cells. Exosomes can contain both pro- and anti-inflammatory cargos but in chronic inflammation, exosomes mostly carry immunosuppressive cargos, e.g. enzymes and miRNAs. The aging process is associated with chronic low-grade inflammation and the accumulation of pro-inflammatory senescent cells into tissues. There is clear evidence that aging increases the number of exosomes in both the circulation and tissues. Especially, the secretion of immunosuppressive exosomes robustly increases from senescent cells. There are observations that the exosomes from senescent cells are involved in the expansion of senescence into neighbouring cells. Interestingly, the age-related exosomes contain immune suppressive cargos which enhance the immunosuppression within recipient immune cells, i.e. tissue-resident and recruited immune cells including M2 macrophages, myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg). It seems that increased immunosuppression with aging impairs the clearance of senescent cells and their accumulation within tissues augments the aging process.     

Role of melanoma inhibitory activity in melanocyte senescence

The protein melanoma inhibitory activity (MIA) is known to be expressed in melanoma and to support melanoma progression. Interestingly, previous studies also observed the expression of MIA in nevi. Concentrating on these findings, we revealed that MIA expression is correlated with a senescent state in melanocytes. Induction of replicative or oncogene‐induced senescence resulted in increased MIA expression in vitro. Notably, MIA knockdown in senescent melanocytes reduced the percentage of senescence‐associated beta‐Gal‐positive cells and enhanced proliferation. Using the melanoma mouse model Tg(Grm1), MIA‐deficient mice supported the impact of MIA on senescence by showing a significantly earlier tumor onset compared to controls. In melanocytes, MIA knockdown led to a downregulation of the cell cycle inhibitor p21 in vitro and in vivo. In contrast, after induction of hTERT in human melanoma cells, p21 regulation by MIA was lost. In summary, our data show for the first time that MIA is a regulator of cellular senescence in human and murine melanocytes.     

Aged‐senescent cells contribute to impaired heart regeneration

Aging leads to increased cellular senescence and is associated with decreased potency of tissue‐specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32–86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.     

Analysis of individual cells identifies cell‐to‐cell variability following induction of cellular senescence

Senescent cells play important roles in both physiological and pathological processes, including cancer and aging. In all cases, however, senescent cells comprise only a small fraction of tissues. Senescent phenotypes have been studied largely in relatively homogeneous populations of cultured cells. In vivo, senescent cells are generally identified by a small number of markers, but whether and how these markers vary among individual cells is unknown. We therefore utilized a combination of single‐cell isolation and a nanofluidic PCR platform to determine the contributions of individual cells to the overall gene expression profile of senescent human fibroblast populations. Individual senescent cells were surprisingly heterogeneous in their gene expression signatures. This cell‐to‐cell variability resulted in a loss of correlation among the expression of several senescence‐associated genes. Many genes encoding senescence‐associated secretory phenotype (SASP) factors, a major contributor to the effects of senescent cells in vivo, showed marked variability with a subset of highly induced genes accounting for the increases observed at the population level. Inflammatory genes in clustered genomic loci showed a greater correlation with senescence compared to nonclustered loci, suggesting that these genes are coregulated by genomic location. Together, these data offer new insights into how genes are regulated in senescent cells and suggest that single markers are inadequate to identify senescent cells in vivo.     

DGCR8‐mediated disruption of miRNA biogenesis induces cellular senescence in primary fibroblasts

The regulation of gene expression by microRNAs (miRNAs) is critical for normal development and physiology. Conversely, miRNA function is frequently impaired in cancer, and other pathologies, either by aberrant expression of individual miRNAs or dysregulation of miRNA synthesis. Here, we have investigated the impact of global disruption of miRNA biogenesis in primary fibroblasts of human or murine origin, through the knockdown of DGCR8, an essential mediator of the synthesis of canonical miRNAs. We find that the inactivation of DGCR8 in these cells results in a dramatic antiproliferative response, with the acquisition of a senescent phenotype. Senescence triggered by DGCR8 loss is accompanied by the upregulation of the cell‐cycle inhibitor p21CIP1. We further show that a subset of senescence‐associated miRNAs with the potential to target p21CIP1 is downregulated during DGCR8‐mediated senescence. Interestingly, the antiproliferative response to miRNA biogenesis disruption is retained in human tumor cells, irrespective of p53 status. In summary, our results show that defective synthesis of canonical microRNAs results in cell‐cycle arrest and cellular senescence in primary fibroblasts mediated by specific miRNAs, and thus identify global miRNA disruption as a novel senescence trigger.     

The bystander effect contributes to the accumulation of senescent cells in vivo

Senescent cells accumulate with age in multiple tissues and may cause age‐associated disease and functional decline. In vitro, senescent cells induce senescence in bystander cells. To see how important this bystander effect may be for accumulation of senescent cells in vivo, we xenotransplanted senescent cells into skeletal muscle and skin of immunocompromised NSG mice. 3 weeks after the last transplantation, mouse dermal fibroblasts and myofibres displayed multiple senescence markers in the vicinity of transplanted senescent cells, but not where non‐senescent or no cells were injected. Adjacent to injected senescent cells, the magnitude of the bystander effect was similar to the increase in senescence markers in myofibres between 8 and 32 months of age. The age‐associated increase of senescence markers in muscle correlated with fibre thinning, a widely used marker of muscle aging and sarcopenia. Senescent cell transplantation resulted in borderline induction of centrally nucleated fibres and no significant thinning, suggesting that myofibre aging might be a delayed consequence of senescence‐like signalling. To assess the relative importance of the bystander effect versus cell‐autonomous senescence, we compared senescent hepatocyte frequencies in livers of wild‐type and NSG mice under ad libitum and dietary restricted feeding. This enabled us to approximate cell‐autonomous and bystander‐driven senescent cell accumulation as well as the impact of immunosurveillance separately. The results suggest a significant impact of the bystander effect for accumulation of senescent hepatocytes in liver and indicate that senostatic interventions like dietary restriction may act as senolytics in immunocompetent animals.     

Cellular senescence contributes to age‐dependent changes in circulating extracellular vesicle cargo and function

Extracellular vesicles (EVs) have emerged as important regulators of inter‐cellular and inter‐organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63⁺ particles mediate these effects. Treatment of macrophages with EV‐like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV‐like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging‐associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.     

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression

Polycystic ovary syndrome (PCOS) is a heterogeneous reproductive disease, characterized by increased ovarian androgen biosynthesis, chronic anovulation and polycystic ovaries. The objective of this study was to identify the altered miRNA expression profiles in follicular fluid derived exosomes isolated from PCOS patients and to investigate the molecular functions of exosomal miR-424-5p. Herein, small RNA sequencing showed that twenty-five miRNAs were differentially expressed between control and PCOS group. The alterations in the miRNA profile were related to the endocrine resistance, cell growth and proliferation, cellular senescence and insulin signaling pathway. Among these differentially expressed miRNAs, we found that the expression of miR-424-5p was significantly decreased in PCOS exosomes and primary granulosa cells (GCs). Exosome-enriched miR-424-5p significantly promoted GCs senescence and suppressed cell proliferation. Similar to the results obtained in the cells transfected with miR-424-5p mimic, miR-424-5p mimic significantly decreased cell proliferation ability and induced senescence, but treatment with miR-424-5p inhibitor got the opposite results. In addition, cell division cycle associated 4 (CDCA4) gene displayed an inverse expression pattern to those of miR-424-5p, was identified as the direct target of miR-424-5p. Overexpression of CDCA4 reversed the effects of exosomal miR-424-5p on GCs via activation of Rb/E2F1 signaling pathway. These results demonstrate that exosomal miR-424-5p inhibits GCs proliferation and induces cellular senescence in PCOS through blocking CDCA4-mediated Rb/E2F1 signaling. Our findings provide new information on abnormal follicular development in PCOS.     

Deep sequencing reveals novel microRNAs and regulation of microRNA expression during cell senescence

In cell senescence, cultured cells cease proliferating and acquire aberrant gene expression patterns. MicroRNAs (miRNAs) modulate gene expression through translational repression or mRNA degradation and have been implicated in senescence. We used deep sequencing to carry out a comprehensive survey of miRNA expression and involvement in cell senescence. Informatic analysis of small RNA sequence datasets from young and senescent IMR90 human fibroblasts identifies many miRNAs that are regulated (either up or down) with cell senescence. Comparison with mRNA expression profiles reveals potential mRNA targets of these senescence-regulated miRNAs. The target mRNAs are enriched for genes involved in biological processes associated with cell senescence. This result greatly extends existing information on the role of miRNAs in cell senescence and is consistent with miRNAs having a causal role in the process.     

Quantitative identification of senescent cells in aging and disease

Senescent cells are present in premalignant lesions and sites of tissue damage and accumulate in tissues with age. In vivo identification, quantification and characterization of senescent cells are challenging tasks that limit our understanding of the role of senescent cells in diseases and aging. Here, we present a new way to precisely quantify and identify senescent cells in tissues on a single‐cell basis. The method combines a senescence‐associated beta‐galactosidase assay with staining of molecular markers for cellular senescence and of cellular identity. By utilizing technology that combines flow cytometry with high‐content image analysis, we were able to quantify senescent cells in tumors, fibrotic tissues, and tissues of aged mice. Our approach also yielded the finding that senescent cells in tissues of aged mice are larger than nonsenescent cells. Thus, this method provides a basis for quantitative assessment of senescent cells and it offers proof of principle for combination of different markers of senescence. It paves the way for screening of senescent cells for identification of new senescence biomarkers, genes that bypass senescence or senolytic compounds that eliminate senescent cells, thus enabling a deeper understanding of the senescent state in vivo.     

Ageing and mesenchymal stem cells derived exosomes: Molecular insight and challenges

Ageing induces a great risk factor that participates in progressing various degenerative diseases morbidities. The main characteristic of ageing is the failure in maintaining homeostasis in the organs with a cellular senescence. Senescence is characterized by reduced cell growth, evade cellular death, and acquiring a senescence‐associated secretory phenotype (SASP). Mesenchymal stem cells (MSCs) are advantageous cells in regenerative medicine, exerting pleiotropic functions by producing soluble factors, such as exosomes. MSCs and their exosomes (MSCs‐Exo) kinetic are affected by ageing and other aged exosomes. Exosomes biogenesis from aged MSCs is accelerated and their exosomal cargoes, such as miRNAs, vary as compared to those of normal cells. Besides, exosomes from aged MSCs loss their regenerative potential and may negatively influence the function of recipient cells. MSCs‐Exo can improve ageing and age‐related diseases; however, the detailed mechanisms remain yet elusive. Although exosomes‐therapy may serve as a new approach to combat ageing, the translation of preclinical results to clinic needs more extensive investigation on exosomes both on their biology and related techniques. Overall, scrutiny on the effect of ageing on MSCs and vice versa is vital for designing novel therapy using MSCs with focus on the management of older individuals.     

Exosomes mediate an epithelial‐mesenchymal transition cascade in retinal pigment epithelial cells: Implications for proliferative vitreoretinopathy

Exosomes have recently emerged as a pivotal mediator of many physiological and pathological processes. However, the role of exosomes in proliferative vitreoretinopathy (PVR) has not been reported. In this study, we aimed to investigate the role of exosomes in PVR. Transforming growth factor beta 2 (TGFß‐2) was used to induce epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, as an in vitro model of PVR. Exosomes from normal and EMTed RPE cells were extracted and identified. We incubated extracted exosomes with recipient RPE cells, and co‐cultured EMTed RPE cells and recipient RPE cells in the presence of the exosome inhibitor GW4869. Both experiments suggested that there are further EMT‐promoting effects of exosomes from EMTed RPE cells. MicroRNA sequencing was also performed to identify the miRNA profiles in exosomes from both groups. We identified 34 differentially expressed exosomal miRNAs (P <. 05). Importantly, miR‐543 was found in exosomes from EMTed RPE cells, and miR‐543‐enriched exosomes significantly induced the EMT of recipient RPE cells. Our study demonstrates that exosomal miRNA is differentially expressed in RPE cells during EMT and that these exosomal miRNAs may play pivotal roles in EMT induction. Our results highlight the importance of exosomes as cellular communicators within the microenvironment of PVR.     

Analysis of deep sequencing exosome-microRNA expression profile derived from CP-II reveals potential role of gga-miRNA-451 in inflammation

Mycoplasma gallisepticum (MG) can cause chronic respiratory disease (CRD) in chickens. While several studies have reported the inflammatory functions of microRNAs during MG infection, the mechanism by which exosomal miRNAs regulate MG-induced inflammation remains to be elucidated. The expression of exosome-microRNA derived from MG-infected chicken type II pneumocytes (CP-II) was screened, and the target genes and function of differentially expressed miRNAs (DEGs) were predicted. To verify the role of exosomal gga-miR-451, Western blot, ELISA and RT-qPCR were used in this study. The results showed that a total of 722 miRNAs were identified from the two exosomal small RNA (sRNA) libraries, and 30 miRNAs (9 up-regulated and 21 down-regulated) were significantly differentially expressed. The target miRNAs were significantly enriched in the treatment group, such as cell cycle, Toll-like receptor signalling pathway and MAPK signalling pathway. The results have also confirmed that gga-miR-451-absent exosomes derived from MG-infected CP-II cells increased inflammatory cytokine production in chicken fibroblast cells (DF-1), and wild-type CP-II cell–derived exosomes displayed protective effects. Collectively, our work suggests that exosomes from MG-infected CP-II cells alter the dynamics of the DF-1 cells, and may contribute to pathology of the MG infection via exosomal gga-miR-451 targeting YWHAZ involving in inflammation.