Optimized protocol for identifying senescence-associated heterochromatin foci (SAHF) as markers of cellular senescence in blood samples

Chromatin remodeling, BRCA1, SAHF and cellular senescence

Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which can stabilize the state of senescence. The retinoblastoma (RB) protein has an important role in SAHF; cells that lack active RB pathway fail to form SAHF. It has been known that the posttranslational modifications of RB, for example, phosphorylation, regulate its function. To date, whether methylation of RB impacts on the SAHF formation is unknown. Here we report that JMJD3, a histone demethylase catalyzing the tri-methylation of H3K27 (H3K27me3), can demethylate the non-histone protein RB at the lysine810 residue (K810), which is a target of the methyltransferase Set7/9. We detected a significant upregulation of JMJD3 during cellular senescence and SAHF formation in WI38 cells induced by H-RasV(12), and we found that ectopic expression of JMJD3 promoted cellular senescence and SAHF formation in WI38 cells. Furthermore, during the process of SAHF assembly, JMJD3 was transported to the cytoplasm and interacted with RB through its demethylase domain JmjC. Significantly, our data demonstrated that the JMJD3-mediated demethylation of RB at K810 impeded the interaction of RB with the protein kinase CDK4 and resulted in reduced level of phosphorylation of RB at Serine807/811 (S807/811), implicating an important role of the interplay between the demethylation and phosphorylation of RB in SAHF assembly. This study highlights the role of JMJD3 as a novel inducer of SAHF formation through demethylating RB and provides new insights into the mechanisms of cellular senescence and SAHF assembly.

In modern pathophysiology and clinical medicine, the search for new, more reliable cellular and molecular biomarkers remains a pressing issue, enabling the assessment of premature aging processes and predicting the risk or severity of many diseases. DNA damage, manifested in particular as micronuclei, is a key factor in initiating the aging process, as it reflects genomic instability. The loss of genomic stability due to physical, chemical, and biological genotoxic factors can lead not only to premature aging of the organism but also to chronic inflammation and the development of neurodegenerative and cardiovascular diseases, diabetes, cancer, and other conditions. Furthermore, studying genomic instability in stem cells is crucial for the development of cellular therapies aimed at slowing aging and treating related diseases. The cytokinesis-block micronucleus assay (CBMN) is proposed as a tool for elucidating the molecular mechanisms linking DNA damage with cellular senescence and aging outcomes at the organismal level. This method provides a range of quantitative indicators for a comprehensive assessment of cellular senescence manifestations, which can be utilized as potential biomarkers for disease risk evaluation and monitoring of disease progression, including as a marker of high cardiovascular risk in patients who have experienced acute cardiac conditions.

Rb Protein is Essential to the Senescence-Associated Heterochromatic Foci Formation Induced by HMGA2 in Primary WI38 Cells

Mesenchymal stem cells (MSCs) have long been viewed as a promising therapeutic for musculoskeletal repair. However, regulatory concerns including tumorgenicity, inconsistencies in preparation techniques, donor-to-donor variability, and the accumulation of senescence during culture expansion have hindered the clinical application of MSCs. Senescence is a driving mechanism for MSC dysfunction with advancing age. Often characterized by increased reactive oxygen species, senescence-associated heterochromatin foci, inflammatory cytokine secretion, and reduced proliferative capacity, senescence directly inhibits MSCs efficacy as a therapeutic for musculoskeletal regeneration. Furthermore, autologous delivery of senescent MSCs can further induce disease and aging progression through the secretion of the senescence-associated secretory phenotype (SASP) and mitigate the regenerative potential of MSCs. To alleviate these issues, the use of senolytic agents to selectively clear senescent cell populations has become popular. However, their benefits to attenuating senescence accumulation in human MSCs during the culture expansion process have not yet been elucidated. To address this, we analyzed markers of senescence during the expansion of human primary adipose-derived stem cells (ADSCs), a population of fat-resident MSCs commonly used in regenerative medicine applications. Next, we used the senolytic agent fisetin to determine if we can reduce these markers of senescence within our culture-expanded ADSC populations. Our results indicate that ADSCs acquire common markers of cellular senescence including increased reactive oxygen species, senescence-associated β-galactosidase, and senescence-associated heterochromatin foci. Furthermore, we found that the senolytic agent fisetin works in a dose-dependent manner and selectively attenuates these markers of senescence while maintaining the differentiation potential of the expanded ADSCs.

Senescence-associated heterochromatin foci (SAHF) are condensed chromatin domains that mark cellular aging. Manual microscopy-based counting of SAHF-positive nuclei is widely used but is labor-intensive, subjective, and limited by binary classification that overlooks nuclear heterogeneity, especially in intermediate senescence or under non-canonical chromatin features. Accurate automated detection remains challenging due to variability in nuclear morphology, fluorescence intensity, and imaging conditions, with current approaches relying on arbitrary thresholds that hinder reproducibility.We present DeepSAHF, a deep learning–based pipeline for robust and reproducible detection of senescence-specific chromatin states from fluorescence microscopy images. The workflow integrates image pre-processing, YOLO-based single nucleus segmentation, and chromatin pattern classification to identify SAHF-positive cells. An extension using a visual language model (VLM) systematically comments on nuclear shape and features, facilitating interpretability and filtering unsuitable nuclei (e.g., out-of-focus). DeepSAHF is trained and tested at 20× resolution on oncogene-induced senescence cells across multiple time points, using curated high-confidence images from literature and novel data to ensure generalization without manual thresholds.By enabling scalable, reproducible, and interpretable analysis of chromatin reorganization, DeepSAHF advances beyond traditional methods, opening avenues to explore nuclear heterogeneity in aging and stress responses, with broad applications in drug screening, regenerative medicine, and cancer biology.

Cellular senescence as a response to multiwalled carbon nanotube (MWCNT) exposure in human mesothelial cells

Pituitary tumor transforming gene (PTTG1, securin) is involved in cell-cycle control through inhibition of sister-chromatid separation. Elevated levels of PTTG1 were found to be associated with many different tumor types that might be involved in late stage tumor progression. However, the role of PTTG1 in early stage of tumorigenesis is unclear. Here we utilized the adenovirus expression system to deliver PTTG1 into normal human fibroblasts to evaluate the role of PTTG1 in tumorigenesis. Expressing PTTG1 in normal human fibroblasts inhibited cell proliferation. Several senescence-associated (SA) phenotypes including increased SA-beta-galactosidase activities, decreased bromodeoxyuridine incorporation, and increased SA-heterochromatin foci formation were also observed in PTTG1-expressing cells, indicating that PTTG1 overexpression induced a senescent phenotype in normal cells. Significantly, the PTTG1-induced senescence is p53-dependent and telomerase-independent, which is distinctively different from that of replicative senescence. The mechanism of PTTG1-induced senescence was also analyzed. Consistent with its role in regulating sister-chromatid separation, overexpression of PTTG1 inhibited the activation of separase. Consequently, the numbers of cells with abnormal nuclei morphologies and chromosome separations were increased, which resulted in activation of the DNA damage response. Thus, we concluded that PTTG1 overexpression in normal human fibroblasts caused chromosome instability, which subsequently induced p53-dependent senescence through activation of DNA-damage response pathway.

DNA-damaging agents are able to induce irreversible cell growth arrest and senescence in some types of tumour cells, thus contributing to the static feature of cancer. However, senescent tumour cells may re-enter the cell cycle, leading to tumour relapse. Understanding the mechanisms that control the viability of senescent cells may be critical for tumour suppression. Primary human fibroblasts undergoing oncogene-induced or replicative senescence are known to form senescence-associated heterochromatin foci (SAHF), which contribute to the stability of the senescent state. However, it is unclear whether SAHF formation is universal in tumour cells. We report that the DNA-damaging agents doxorubicin and 7-ethyl-10-hydroxycamptothecin were able to induce the formation of SAHF in some tumour cell types, and this induction was accompanied by activation of the retinoblastoma protein pathway. By contrast, tumour cells in which the retinoblastoma protein pathway could not be activated by doxorubicin or 7-ethyl-10-hydroxycamptothecin failed to form SAHF. In parallel, tumour cells with deficient retinoblastoma protein were also unable to form SAHF. In addition, we show that the mitogen-activated protein kinase p38 pathway was involved in tumour cell SAHF formation in response to doxorubicin and 7-ethyl-10-hydroxycamptothecin. Furthermore, HMG box transcription factor 1 (HBP1), a downstream target of the mitogen-activated protein kinase p38-mediated senescence pathway, was required for SAHF formation. Taken together, the results of the present study highlight the roles of the mitogen-activated protein kinase p38/retinoblastoma protein pathway in tumour cell SAHF formation in response to DNA-damaging agents, and provide new insights into the mechanisms of DNA damage-mediated tumour suppression.

Cardiovascular diseases are the most common complications of type 2 diabetes mellitus (T2DM) and remain the main cause of mortality in this category of patients. Currently, there is more and more data that confirm the influence of nutritional factors not only on the achievement of target values of metabolic parameters in diabetes, but also on predictors of cardiovascular risk, and also demonstrate their role as independent predictors. In this regard, the study of the role of nutritional factors in the formation of high cardiovascular risk in patients with T2DM is of high practical importance and relevance. The aim was to assess the role of nutritional factors in the formation of high total cardiovascular risk in patients with T2DM. Material and methods. An open clinical controlled prospective observation was carried out for one year in 110 patients with T2DM (87 women and 23 men, mean age 56.7±8.6 years). The average value of glycated hemoglobin (HbA1c) in the group was 8.95±2.09%, the av erage value of the body mass index was 32.3±6.2 kg/m2. All patients were assessed for traditional cardiovascular risk factors, markers of systemic inflammation and endothelial dysfunction, nutritional status was assessed by analyzing the frequency of consumption and a general semi-quantitative assessment of the intake of nutrients, food groups, and energy. During a one-year prospective follow-up, total cardiovascular endpoints were recorded and the effect of patient education on metabolic parameters, nutritional factors, and cardiovascular risk was assessed. Results. In patients with T2DM who had various clinical forms of cardiovascular diseases, the diet had an increased level of fats, which exceeded the intake in individuals without complications (p=0.013), and saturated fatty acids (SFA) (p=0.003). The risk of developing cardiovascular events in patients with T2DM increased by 5 times under excessive consumption of products containing SFA (meat, animal fats, sausages) (OR 5.34; CI 3.05-10.22, p=0.001). The decrease in body weight in the target range (by 7-10% during the year) was characterized by a decrease in the HbA1c by 11.9%, postprandial glycemia by 25.7%, total cholesterol by 20.4%, atherogenic coefficient by 25.0%, plasminogen activator inhibitor-1 by 8.5%, highly sensitive C-reactive protein by 27.4%, systolic blood pressure by 6.9%, and also was accompanied by significant decrease in unfavorable total cardiovascular events during the year (p=0.024). In addition, in the group of patients who underwent training, there was a decrease in the total amount of calories consumed (p=0.018), consumption of SFA (p=0.021) and mono- and disaccharides (p=0.001), an increase in dietary fiber in the diet (p=0.015). Conclusion. In the course of the study, an imbalance in the nutritional components of the diet in patients with T2DM was revealed. The significant role of alimentary factors in the formation of high cardiovascular risk has been demonstrated, as well as the effectiveness of therapeutic education of patients in terms of managing behavioral risk factors.

Osteoprotegerin (OPG), a member of the tumour necrosis factor receptor (TNFR) superfamily of proteins known to be involved in a large number of biological systems, plays a pivotal role in bone remodelling. In addition to the roles of OPG in bone metabolism, it has been reported to be associated with a high cardiovascular risk in patients with metabolic syndrome. In most cases, the exact functions of OPG remain to be established; however, the widespread expression of OPG suggests that this molecule may have multiple biological activities, mainly in the cardiometabolic environment. The aim of this study was to evaluate the value of OPG as a predictive marker for cardiovascular and metabolic risk in osteoporotic patients. The study group comprised patients with osteoporosis, in order to evaluate the association between OPG serum levels and cardiovascular pathology. Our results revealed significant correlations between classical biochemical bone and metabolic parameters, such as osteocalcin and parathyroid hormone with lipid and glucose biomarkers, sustaining the crosstalk between calcium and bone parameters and cardiovascular risk. The OPG serum level proved to have a significant and independent predictive value for metabolic syndrome (MetS) as a cardiovascular risk standard in osteoporotic patients. The OPG serum levels were increased in patients with MetS as a protective response against the atherosclerotic lesions. The serum levels of 25-hydroxy vitamin D had significant and independent predictive value for cardiovascular and metabolic risk in our subjects, sustaining the active role of vitamin D beyond the area of bone metabolism.

Our goal is to understand the impact of chromatin structure on cell proliferation, cell and tissue aging, cancer and cancer therapies. To this end, we have investigated the formation of specialized domains of facultative heterochromatin, called Senescence Associated Heterochromatin Foci (SAHF), in senescent human cells. A complex of histone chaperones, HIRA and ASF1a, drives formation of SAHF. Remarkably, although SAHF are highly compacted domains of heterochromatin, these domains of facultative heterochromatin largely exclude other domains of chromatin at telomeres and pericentromeres, which are themselves thought to be constitutively heterochromatic. The relationship between SAHF formation and these other domains of heterochromatin is discussed. Also, in the course of our studies, we have obtained evidence that points to a novel function for the widely-studied but poorly-understood family of heterochromatin proteins, HP1 proteins. We propose that HP1 proteins are essential components of a dynamic nuclear response that senses and rectifies defects in epigenetic information, encoded in chromatin through histone modifications and DNA methylation. We further propose that defects in this essential "chromatin repair" response in transformed human cells contributes to the preferential killing of cancer cells by the epigenetic cancer therapies that are currently in clinical development.

Introduction: Advanced oxidation protein products (AOPPs), the novel marker of oxidative stress, have been found to be elevated in preeclampsia (PE). To date, the effect of AOPPs on the senescence of trophoblast cells is still unclear. In this study, we investigated whether AOPPs promoted the senescence of trophoblast cells and explored the underlying mechanisms of AOPPs-induced aging process which may facilitate the progression of PE. Methods: The trophoblast cell line HTR-8/SV neo cells were cultured in the presence of PBS, AOPPs, AOPPs plus an anti-oxidant N-acetyl-L-cysteine (NAC). In some experiments, cells were pre-treated with rapamycin (an activator of autophagy), 3-MA (an inhibitor of autophagy), or cyclic pifithrin-α (PFT-α, an antagonist of p53), and then treated with AOPPs. Cellular senescence was analyzed by measuring the levels of senescence-associated β-galactosidase (SA β-Gal), senescence-associated heterochromatin foci (SAHF), mitochondrial membrane potential (ΔΨm), and cell cycle. Cell autophagic flux was analyzed by measuring tandem fluorescence-tagged LC3 reporter (mCherry-EGFP-LC3). Levels of p53, phosphorylated p53 (p-p53), p21, BECN1, p62, p-mTOR and p-p70S6K were measured by western blot. Results: Treatment with AOPPs significantly increased the levels of SA β-Gal and SAHF, the percentage of cells in the G0/G1 phase, and decreased cell ΔΨm compared with the control group. Co-treatment with NAC and AOPPs significantly reversed AOPPs-induced senescence. Pre-treatment with rapamycin or 3-MA significantly inhibited or promoted AOPPs-induced senescence, respectively. In addition, administration of AOPPs significantly decreased the numbers of mCherry+EGFP+ autophagosomes and mCherry+EGFP- autolysosomes in cells compared with cells treated with PBS. Furthermore, AOPPs significantly increased the levels of proteins p-p53, p21, p-mTOR and p-p70S6K compared with the control group. Pre-treatment with rapamycin or PFT-α significantly down-regulated the levels of SA β-Gal, SAHF, p-p53, p21, autophagy related protein p62, the percentage of cells in the G0/G1 phase, and significantly up-regulated ΔΨm, autophagy related protein BECN1, autophagosomes and autolysosomes compared with cells only treated with AOPPs. Conclusion: AOPPs may induce trophoblast cell senescence by inhibiting the autophagy process in a p53/mTOR/p70S6K-dependent pathway.

Downregulation of Wnt Signaling Is a Trigger for Formation of Facultative Heterochromatin and Onset of Cell Senescence in Primary Human Cells

Background Guidelines recommend intensive low-density-lipoprotein cholesterol (LDL-C) lowering in high cardiovascular (CV) risk patients with acute coronary syndrome (ACS), stroke and critical limb ischemia (CLI). Purpose We evaluated LDL-C goal attainment and lipid-lowering treatment (LLT) in a Chinese population with ACS, stroke and CLI patients. Methods We retrospectively evaluated consecutive high CV risk patients discharged between 2013 and 2017 from 3 hospitals in Hong Kong. Lipid profile and LLT were compared among 3 patient groups: ACS, Stroke and CLI. Results Of 10,168 high-CV risk patients (mean age 70.6±13.7 years; 62.4% male), 64.0% were ACS, 33.6% stroke and 2.5% CLI. Between baseline and 12-month, mean LDL-C reduced from 2.7±1.1 to 2.0±0.8 mmol/L in ACS patients, 2.7±1.0 to 2.0±0.7 mmol/L in stroke patients and 2.5±1.0 to 2.2±0.9 mmol/L in CLI patients (p<0.01). Proportion of CLI patients (29.9%) who achieved target LDL-C <1.8mmol/L at month 12 was significantly lower than stroke (45.6%) and ACS (48.2%) patients (p<0.01). The mean residual distance to target LDL-C was greatest in CLI (0.8±0.8 mmol/L) compared to stroke (0.6±0.6 mmol/L) and ACS (0.7±0.7 mmol/L) patients (p<0.01). Use of statin therapy on discharge was highest in ACS (88.4%) compared to stroke (78.3%) and CLI (52.6%) patients (p<0.01). But use of high-potency statin (rosuvastatin ≥20mg, atorvastatin ≥40mg or simvastatin ≥80mg) on discharge was very low in stroke (3.0%) and CLI (2.0%) compared to ACS (21.4%, p<0.01) patients. At 12 months 28.8% ACS, 34.3% stroke and 51.4% CLI patients were on no LLT (p<0.01) and the use of high-potency statin did not change significantly (3.0% in stroke, p=0.99; and 1.2% in CLI, p=0.48). Despite the poor achievement in LDL-C target in CLI patients, the proportion of CLI patients switching to high-potency statin (0.8%) was significantly lower than stroke (1.3%) and ACS (5.2%) patients (p<0.01). Conclusion This study demonstrated significant therapeutic gaps in lipid-lowering management in high CV risk patients. In particular, CLI patients were less aggressively treated with LLT and hence larger proportion of patient not achieving LDL-C target compared to ACS and stroke patients.