Β-galactosidase Positive Cells Research Articles

A squalene analog 4,4′-diapophytofluene from coconut leaves having antioxidant and anti-senescence potentialities toward human fibroblasts and keratinocytes

Coconut (Cocos nucifera) leaves, an unutilized resource, enriched with valuable bioactive compounds. Spectral analysis of purified pentane fraction of coconut leaves revealed the presence of a squalene analog named 4,4′-diapophytofluene or in short 4,4′-DPE (C30H46). Pure squalene standard (PSQ) showed cytotoxicity after 8 µg/ml concentration whereas 4,4′-DPE exhibited no cytotoxic effects up to 16 µg/ml concentration. On senescence-induced WI38 cells, 4,4′-DPE displayed better percentage of cell viability (164.5% at 24 h, 159.4% at 48 h and 148% at 72 h) compared to PSQ and BSQ (bio-source squalene) with same time duration. Similar trend of result was found in HaCaT cells. SA-β-gal assay showed that number of β-galactosidase positive cells were significantly decreased in senescent cells (WI38 and HaCaT) after treated with 4,4′-DPE than PSQ, BSQ. Percentage of ROS was increased to 60% in WI38 cells after olaparib treatment. When PSQ, BSQ and 4,4′-DPE were applied separately on these oxidative-stress-induced cells for 48 h, the overall percentage of ROS was decreased to 39.3%, 45.6% and 19.3% respectively. This 4,4′-DPE was found to be more effective in inhibiting senescence by removing ROS as compared to squalene. Therefore, this 4,4′-DPE would be new potent senotherapeutic agent for pharmaceuticals and dermatological products.

Gastrodin alleviates premature senescence of vascular endothelial cells by enhancing the Nrf2/HO-1 signalling pathway.

Endothelial dysfunction is an independent risk factor for stroke. The dysfunction of endothelial cells (EC) is closely concerned with EC senescence. Gastrodin (GAS) is an organic compound extracted from the dried root mass of the Orchidaceae plant Gastrodiae gastrodiae. It is used clinically to treat diseases such as vertebrobasilar insufficiency, vestibular neuronitis and vertigo. In the present study, we used hydrogen peroxide (H2 O2 )-induced human umbilical vein endothelial cells (HUVECs) to establish an invitro EC senescence model and to investigate the role and mechanism of GAS in EC senescence. It's found that H2 O2 -treated HUVECs increased the proportion of senescence-associated β-galactosidase (SA β-gal) positive cells and the relative protein expression levels of senescence-associated cyclin p16 and p21. In addition, GAS reduced the proportion of SA β-gal positive cells and the relative protein expression levels of p16 and p21, and increased the proliferation and migration ability of HUVECs. Meanwhile, GAS increased the expression of the anti-oxidative stress protein HO-1 and its nuclear expression level of Nrf2. The anti-senescence effect of GAS was blocked when HO-1 expression was inhibited by SnPPIX. Furthermore, absence of HO-1 abolished the effect of GAS on HUVEC proliferation and migration. In conclusion, GAS ameliorated H2 O2 -induced cellular senescence and enhanced cell proliferation and migration by enhancing Nrf2/HO-1 signalling in HUVECs. These findings of our study expanded the understanding of GAS pharmacology and suggested that GAS may offer a potential therapeutic agent for stroke.

Nervonic Acid Inhibits Replicative Senescence of Human Wharton's Jelly-Derived Mesenchymal Stem Cells.

Cellular senescence causes cell cycle arrest and promotes permanent cessation of proliferation. Since the senescence of mesenchymal stem cells (MSCs) reduces proliferation and multipotency and increases immunogenicity, aged MSCs are not suitable for cell therapy. Therefore, it is important to inhibit cellular senescence in MSCs. It has recently been reported that metabolites can control aging diseases. Therefore, we aimed to identify novel metabolites that regulate the replicative senescence in MSCs. Using a fecal metabolites library, we identified nervonic acid (NA) as a candidate metabolite for replicative senescence regulation. In replicative senescent MSCs, NA reduced senescence-associated β-galactosidase positive cells, the expression of senescence-related genes, as well as increased stemness and adipogenesis. Moreover, in non-senescent MSCs, NA treatment delayed senescence caused by sequential subculture and promoted proliferation. We confirmed, for the first time, that NA delayed and inhibited cellular senescence. Considering optimal concentration, duration, and timing of drug treatment, NA is a novel potential metabolite that can be used in the development of technologies that regulate cellular senescence.

Ebselen, Iron Uptake Inhibitor, Alleviates Iron Overload-Induced Senescence-Like Neuronal Cells SH-SY5Y via Suppressing the mTORC1 Signaling Pathway.

Increasing evidence highlights that excessive iron accumulation in the brain plays a vital role in neuronal senescence and is implicated in the pathogenesis of age-related neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD). Therefore, the chemical compounds that eliminate an iron overload may provide better protection against oxidative stress conditions that cause the accumulation of senescent cells during brain aging. Ebselen has been identified as a strongly useful compound in the research on redox biology mechanisms. We hypothesized that ebselen could alleviate an iron overload-induced oxidative stress and consequently reverses the senescence-like phenotypes in the neuronal cells. In the present study, SH-SY5Y cells were treated with ferric ammonium citrate (FAC) before ebselen, and the evaluation of the cellular iron homeostasis, the indicators of oxidative stress, and the onset of senescence phenotypes and mechanisms were carried out accordingly. Our findings showed that ebselen ameliorated the FAC-mediated iron overload by decreasing the expression of divalent metal transporter 1 (DMT1) and ferritin light chain (FT-L) proteins. In contrast, it increased the expression of ferroportin 1 (FPN1) protein and its correlation led to a decrease in the expression of the cytosolic labile iron pool (LIP). Furthermore, ebselen significantly reduced reactive oxygen species (ROS) and rescued the mitochondrial membrane potential (ΔΨm). Notably, ebselen restored the biomarkers of cellular senescence by reducing the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and senescence-associated secretory phenotypes (SASP). This also suppressed the expression of p53 protein targeting DNA damage response (DDR)/p21 cyclin-dependent kinase (CDK) inhibitor through a mTORC1 signaling pathway. Potentially, ebselen could be a therapeutic agent for treating brain aging and AD by mitigating iron accumulation and restoring senescence in SH-SY5Y cells.

Persistent Hypoxia with Intermittent Aggravation Causes Imbalance in Smad3/Myocardin-Related Transcription Factor Signaling with Consequent Endothelial Senescence and Pulmonary Arterial Remodeling.

Loss of Smad3 and the consequent activation of myocardin-related transcription factor (MRTF) are associated with vascular pathologies. This study aimed to examine the impact of persistent hypoxia with intermittent aggravation (PI hypoxia) on cellular senescence and pulmonary arterial remodeling mediated by the Smad3/MRTF imbalance. We examined the effects of PI hypoxia on the Smad3/MRTF pathway and cellular senescence using human pulmonary artery endothelial cells (HPAECs) and in vivo studies in rats. The senescent degree was evaluated using β-galactosidase staining, p16 quantitation and the measurement of senescence-associated secretory phenotype. Structural data in the pathological analysis of pulmonary artery remodeling were collected. Compared to the control, HPAECs and pulmonary tissue from rats exposed to PI hypoxia showed a significantly higher senescent degree, lower expression of Smad3, and higher MRTF levels. The overexpression of Smad3 significantly mitigated HPAECs senescence in vitro. Further, treatment with CCG-203971, which inhibits MRTF, increased Smad3 levels and reduced β-galactosidase positive cells in rat lung tissue. This intervention also alleviated PI hypoxia-induced pathological changes, including remodeling indices of pulmonary arterial thickening, muscularization, and collagen formation. In conclusion, imbalanced Smad3/MRTF signaling is linked to PI hypoxia-induced senescence and pulmonary arterial remodeling, making it a potential therapeutic target for patients with sleep apnea and chronic obstructive pulmonary disease.

Senomorphic effect of diphenyleneiodonium through AMPK/MFF/DRP1 mediated mitochondrial fission

With an aging population and the numerous health impacts associated with old age, the identification of anti-aging drugs has become an important new research direction. Although mitochondria have been recognized to affect aging, anti-aging drugs specifically targeting the mitochondria are less well characterized. In this study, diphenyleneiodonium (DPI) was identified as a potential senomorphic drug that functions by promoting mitochondrial fission. DPI significantly reduced the number of senescence-associated β-galactosidase (SA-β-gal) positive cells and increased the number of proliferating Ki-67 positive cells in BrdU or irradiation stress-induced senescent NIH3T3 cells or IMR90 cells and mouse embryonic fibroblasts (MEFs) replicative senescent cells. Cell cycle arrest genes and senescence-associated secretory phenotype (SASP) factors were downregulated with DPI treatment. In addition, the oxygen consumption rate (OCR) of mitochondrial respiration showed that DPI significantly reduced senescence-associated hyper OCR. Mechanistically, DPI promoted mitochondrial fission by enhancing AMPK/MFF phosphorylation and DRP1 mitochondrial translocation. Inhibition of DRP1 by Mdivi-1 abolished DPI-induced mitochondrial fission and the anti-senescence phenotype. Importantly, Eighty-eight-week-old mice treated with DPI had significantly reduced numbers of SA-β-gal positive cells and reduced expression of cell cycle arrest genes and SASP factors in their livers and kidneys. Pathological and functional assays showed DPI treatment not only reduced liver fibrosis and immune cell infiltration but also improved aged-related physical impairments in aged mice. Taken together, our study identified a potential anti-aging compound that exerts its effects through modulation of mitochondrial morphology.

Dopamine D1 receptor activation ameliorates ox-LDL-induced endothelial cell senescence via CREB/Nrf2 pathway

Endothelial cell senescence is involved in endothelial dysfunction and aging-related vascular diseases. The D1-like dopamine receptor (DR1), a number of G-protein-coupled receptors, is currently under consideration as a potential therapeutic target for the prevention of atherosclerosis. However, the role of DR1 in regulating ox-LDL-stimulated endothelial cell senescence remains unknown. Here, we found that the elevated Prx hyperoxidation and reactive oxygen species (ROS) levels in ox-LDL-treated Human umbilical vein endothelial cells (HUVECs) were observed, suppressed by DR1 agonist SKF38393. Increased proportion of senescence-associated β-galactosidase (SA-β-gal) positive staining cells and activated p16/p21/p53 pathway in ox-LDL-treated HUVECs were significantly abolished by DR1 activation. In addition, SKF38393 increased the phosphorylation of cAMP response element-binding protein (CREB) at serine-133, nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and expression of HO-1 in HUVECs. In contrast, adding H-89, a PKA inhibitor, diminished the effects of DR1 activation. Further studies performed with DR1 siRNA confirmed that DR1 was involved in CREB/Nrf2 pathway. Taken together, DR1 activation reduces ROS production and cell senescence by upregulating CREB/Nrf2 antioxidant signaling in ox-LDL-induced endothelial cells. Thus, DR1 could be a potential molecular target to counteract oxidative stress-induced cellular senescence.

Abstract P1-10-09: AQP4 inhibition prevents cytotoxic edema of AQP4+ astrocytes but promotes tumor growth of AQP4+ breast cancer brain metastasis

Abstract Brain edema is a complication of radiation used to treat brain metastasis (BM) in which the brain parenchyma accumulates fluid and ions, often leading to the suspension of systemic anticancer treatment. While brain edema is often attributed to disruption of the Blood Brain Barrier (BBB), RTx induces cytotoxic-edema, a premorbid cellular process whereby extracellular Na+ and other cations enter into neurons and astrocytes and accumulate intracellularly, resulting in osmotic expansion of the cells and necrotic cell death. Aquaporin 4 (AQP4) is a main regulator of osmotic expansion (water intake) in astrocytes and we have shown that RTx upregulates AQP4 in astrocytes and leads to astrocytic swelling in vitro. However, whether pharmacological modulation of AQP4 could be used to prevent cytotoxic brain edema in RTx-treated BM and its impact on metastatic tumor progression remains unknown. Goal: To determine if the FDA-approved drug Topiramate (TPM), an anti-epileptic drug able to inhibit AQP4) can prevent astrocytic swelling in vitro, reduce RTx-induced brain edema and modulate brain metastatic progression. Results: Electron microscopy of brain cortex from mice treated with 35 Gy RTx showed acute astrocytic end-feet swelling and increase in AQP4 expression compared with non-irradiated mice. A single 8 Gy dose increased astrocytic cell area of human astrocytes by 4.8 fold compared with non-irradiated cells 24 h after RTx. This increased cell-swelling did not result from senescence-associated cellular hypertrophy, as staining of senescent β-galactosidase positive (SA-β-Gal+) cells showed that Rtx-induced astrocytic area only increased significantly in non-senescent (SA-β-Gal- cells). shAQP4s reduced AQP4 levels by 60% and 50%, respectively, and significantly reduced RTx-induced astrocytic swelling. Since there are no FDA-approved AQP4 inhibitors, we tested whether the AQP4-blocking function of TPM could be sufficient to prevent cytotoxic edema, prevent BBB dysfunction and protect from necrotic cell death in vitro. TPM pretreatment did not alter radiation-induced ERK1/2 or AKT activation (a known maker of radioprotection) in astrocytes, but TPM decreased radiation-induced PARP-cleavage, pP38 and pJNK levels. TPM prevented loss of Trans-electric epithelial resistance (TEER) of Rtx-treated astrocytes, but was not able to protect astrocytes from ultimate cell death. Immunohistochemical analysis of a cohort of breast cancer BM showed heterogeneous AQP4 expression in cancer cells ranging from 1.6% to 91% AQP4+ tumoral areas and from 0.6% to 86.9% in stroma. AQP4 inhibition using shRNAs decreased proliferation and survival of AQP4 + 231BR, and EO711 cells in vitro. However, TPM did not alter survival of AQP4+ or AQP4- cells in vitro, suggesting that while AQP4 expression is important for survival of AQP4+ cells in vitro, the inhibition of AQP4 function by TPM is not sufficient to decrease their growth. To determine if TPM could decrease brain edema without negatively impacting tumor progression, female NSG mice were injected intracardially with JmT1BR3 AQP4-cells and ten days later randomized to (1) RTx + vehicle, (2) RTx + TPM (2 days prior to irradiation), (3) Non-RTx + vehicle, and (4) Non-RTx + TPM. TPM decreased brain-water content (a marker of brain edema) in irradiated mice as compared with vehicle-treated mice, without alteration of metastatic burden 21 days post-injection. However, a similar study using AQP4+ E0711 cells in C57Bl6 mice showed TPM was less effective in decreasing brain water content and resulted in a significant increase in extracranial metastatic tumor burden, suggesting that TPM can promote tumor progression by non-tumor intrinsic mechanisms. Conclusions: while TPM shows promise in preventing RTx-induced brain edema, our results show a potential pro-tumorigenic mechanism for TPM that warrants further investigation. Citation Format: Maria J. Contreras-Zarate, Karen ALvarez-Eraso, Nicole Tsuji, Peter Kabos, D.Ryan Ormond, Sana Karam, Diana Cittelly. AQP4 inhibition prevents cytotoxic edema of AQP4+ astrocytes but promotes tumor growth of AQP4+ breast cancer brain metastasis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-10-09.

Ethanol-induced conditioned taste aversion and associated neural activation in male rats: Impact of age and adolescent intermittent ethanol exposure.

Individuals that initiate alcohol use at younger ages and binge drink during adolescence are more susceptible to developing alcohol use disorder. Adolescents are relatively insensitive to the aversive effects of alcohol and tend to consume significantly more alcohol per occasion than adults, an effect that is conserved in rodent models. Adolescent typical insensitivity to the aversive effects of alcohol may promote greater alcohol intake by attenuating internal cues that curb its consumption. Attenuated sensitivity to the aversive effects of alcohol is also retained into adulthood following protracted abstinence from adolescent intermittent ethanol (AIE) exposure. Despite these effects, much remains unknown regarding the neural contributors. In the present study, we used a conditioned taste aversion (CTA) paradigm to investigate neuronal activation in late-developing forebrain structures of male adolescents and adult cFos-LacZ transgenic rats as well as in AIE adults following consumption of 0.9% sodium chloride previously paired with an intraperitoneal injection of 0, 1.5 or 2.5 g/kg of ethanol. Adults that were non-manipulated or received water exposure during adolescence showed CTA to both ethanol doses, whereas adolescents displayed CTA only to the 2.5 g/kg ethanol dose. Adults who experienced AIE did not show CTA. Adults displayed increased neuronal activation indexed via number of β-galactosidase positive (β-gal+) cells in the prefrontal and insular cortex that was absent in adolescents, whereas adolescents but not adults had a reduced number of β-gal+ cells in the central amygdala. Adults also displayed greater cortical-insular functional connectivity than adolescents as well as insular-amygdalar and prefrontal cortex-accumbens core functional connectivity. Like adolescents, adults previously exposed to AIE displayed reduced prefrontal-insular cortex and prefrontal-accumbal core functional connectivity. Taken together, these results suggest that attenuated sensitivity to the aversive effects of ethanol is related to a loss of an insular-prefrontal cortex-accumbens core circuit.

Focal adhesion alterations in G0-positive melanoma cells.

Melanoma is a highly heterogeneous malignant tumor that exhibits various forms of drug resistance. Recently, reversal transition of cancer cells to the G0 phase of the cell cycle under the influence of therapeutic drugs has been identified as an event associated with tumor dissemination. In the present study, we investigated the ability of chemotherapeutic agent dacarbazine to induce a transition of melanoma cells to the G0 phase as a mechanism of chemoresistance. We used the flow cytometry to analyze cell distribution within cell cycle phases after dacarbazine treatment as well as to identifyG0 -positive cells population. Transcriptome profiling was provided to determine genes associated with dacarbazine resistance. We evaluated the activity of β-galactosidase in cells treated with dacarbazine by substrate hydrolysis. Cell adhesion strength was measured by centrifugal assay application with subsequent staining of adhesive cells with Ki-67 monoclonal antibodies. Ability of melanoma cells to metabolize dacarbazine was determined by expressional analysis of CYP1A1, CYP1A2, CYP2E1 followed by CYP1A1 protein level evaluation by the ELISA method. The present study determined that dacarbazine treatment of melanoma cells could induce an increase in the percentage of cells in G0 phase without alterations of β-galactosidase positive cells which corresponded to the fraction of the senescent cells. Transcriptomic profiling of cells under dacarbazine induction of G0 -positive cells percentage revealed that 'VEGFA-VEGFR2 signaling pathway' and 'Cell cycle' signaling were mostly enriched by dysregulated genes. 'Focal adhesion' signaling was also found to be triggered by dacarbazine. In melanoma cells treated with dacarbazine, an increase in G0 -positive cells among adherent cells was found. Dacarbazine induces the alteration in a percentage of melanoma cells residing in G0 phase of a cell cycle. The altered adhesive phenotype of cancer cells under transition in the G0 phase may refer to a specific intercellular communication pattern of quiescent/senescent cancer cells.

Senescence Induced by UVB in Keratinocytes Impairs Amino Acids Balance

Skin is one of the most exposed organs to external stress. Namely, UV rays are the most harmful stress that could induce important damage leading to skin aging and cancers. At the cellular level, senescence is observed in several skin cell types and contributes to skin aging. However, the origin of skin senescent cells is still unclear but is probably related to exposure to stresses. In this work, we developed an invitro model of UVB-induced premature senescence in normal human epidermal keratinocytes. UVB-induced senescent keratinocytes display a common senescent phenotype resulting in an irreversible cell cycle arrest, an increase in the proportion of senescence-associated β-galactosidase‒positive cells, unrepaired DNA damage, and a long-term DNA damage response activation. Moreover, UVB-induced senescent keratinocytes secrete senescence-associated secretory phenotype factors that influence cutaneous squamous cell carcinoma cell migration. Finally, a global transcriptomic study highlighted that senescent keratinocytes present a decrease in the expression of several amino acid transporters, which is associated with reduced intracellular levels of glycine, alanine, and leucine. Interestingly, the chemical inhibition of the glycine transporter SLC6A9/Glyt1 triggers senescence features.

Proteomic profiling reveals neuronal ion channel dysregulation and cellular responses to DNA damage-induced cell cycle arrest and senescence in human neuroblastoma SH-SY5Y cells exposed to cypermethrin

Cypermethrin (CYP), a synthetic pyrethroid of class II, is widely used as a pesticide worldwide. The primary target of cypermethrin is a voltage-gated sodium channel. The neurotoxicity of CYP has been extensively studied in terms of affecting neuronal development, increasing cellular oxidative stress, and apoptosis. However, little is known about how it affects the expression of channel proteins involved in synaptic transmission, as well as the effects of cypermethrin on DNA damage and cell cycle processes. We found that the ligand and voltage-gated calcium channels and proteins involved in synaptic transmission including NMDA 1 receptor subunit, alpha 1A-voltage-dependent calcium channel, synaptotagmin-17, and synaptojanin-2 were downregulated in CYP-treated cells. After 48 h of CYP exposure, cell viability was reduced with flattened and enlarged morphology. The levels of 23 proteins regulating cell cycle processes were altered in CYP-treated cells, according to a proteomic study. The cell cycle analysis showed elevated G0/G1 cell cycle arrest and DNA fragmentation at the sub-G0 stage after CYP exposure. CYP treatment also increased senescence-associated β-galactosidase positive cells, DNA damage, and apoptotic markers. Taken together, the current study showed that cypermethrin exposure caused DNA damage and hastened cellular senescence and apoptosis via disrupting cell cycle regulation. In addition, despite its primary target sodium channel, CYP might cause synaptic dysfunction via the downregulation of synaptic proteins and dysregulation of synapse-associated ion channels.

Insulin-like growth factor binding protein 5 accelerate the senescence of periodontal ligament stem cells.

Evidences have showed stem cell mediated tissue regeneration is a promising method for the treatment of periodontitis. Insulin-like growth factor binding proteins-5 (IGFBP5) is a member of the insulin growth factor (IGFs) family and plays a regulatory role in cell proliferation and differentiation. Our previous study showed that IGFBP5 can promote osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and enhance periodontal tissue regeneration mediated by PDLSCs. However, the function of IGFBP5 in the process of PDLSCs senescence remains unclear. The present study showed IGFBP5 mRNA level was highly expressed in passage-induced aged PDLSCs cells. IGFBP5 knockdown decreased the ratio of senescence associated β-galactosidase (SA-β-Gal) positive cells, enhanced the activity of TERT, and down-regulated the expression levels of P16, P21, P53 mRNA and protein. Overexpression of IGFBP5 increased the ratio of SA-β-Gal positive staining PDLSCs, decreased the activity of telomerase TERT, and up-regulated the expression levels of P16, P21, P53 mRNA and protein related to PDLSCs senescence. In conclusion, IGFBP5 can accelerate the senescence of PDLSCs, indicating the potential target for maintaining the "young state" of stem cells.

Vascular senescence in progeria: role of endothelial dysfunction.

AimsHutchinson–Gilford progeria syndrome (HGPS) is a pre-mature aging disorder caused by the mutation of the LMNA gene leading to an irreversibly farnesylated lamin A protein: progerin. The major causes of death in HGPS are coronary and arterial occlusive disease. In the murine model of HGPS, vascular smooth muscle cell (VSMC) loss is the primary vascular manifestation, which is different from the arterial occlusive disease seen in older patients.Methods and resultsTo identify the mechanisms of HGPS vascular disease in humans, we differentiated isogenic endothelial cells (ECs) and VSMCs from HGPS-induced pluripotent stem cells (iPSCs) and control-iPSCs. Both HGPS-ECs and HGPS-VSMCs manifested cellular hallmarks of aging, including dysmorphic nuclei, impaired proliferation, increased β-galactosidase staining, shortened telomeres, up-regulated secretion of inflammatory cytokines, increased DNA damage, loss of heterochromatin, and altered shelterin protein complex (SPC) expression. However, at similar days after differentiation, even with lower levels of progerin, HGPS-ECs manifested more severe signs of senescence, as indicated in part by a higher percentage of β-galactosidase positive cells, shorter telomere length, and more DNA damage signals. We observed increased γH2A.X binding to RAP1 and reduced TRF2 binding to lamin A in HGPS-ECs but not in HGPS-VSMCs. The expression of γH2A.X was greater in HGPS-ECs than in HGPS-VSMCs and is associated with greater telomere shortening, impaired SPC interactions, and loss of heterochromatin.ConclusionAlthough progerin expression has a deleterious effect on both ECs and VSMCs, the dysfunction is greater in HGPS-ECs compared with HGPS-VSMCs. This study suggests that an endothelial-targeted therapy may be useful for HGPS patients.

Abstract 1694: Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells

Abstract Introduction: Detection of circulating free DNA (cfDNA) is a promising tool to monitor and predict tumor progression and treatment efficacy. Our group has shown the clinical relevance of cfDNA as a biomarker of tumor development and progression in several cancer models, including colorectal carcinoma and melanoma. cfDNA may stem from a combination of apoptosis, necrosis, and cellular secretions. However, the source and mechanism of cfDNA release in cancer cells are still unclear. Indeed, while some data state that cfDNA is derived from apoptosis, other studies report it is mainly a product of cellular secretions. To better understand cfDNA release and its correlation with tumor biology, this study aimed to assess cfDNA kinetics under cell death, cell cycle arrest, and senescence by using an in vitro cancer model. Methods: Colorectal (HTC116 and HT29), lung (A549), and melanoma (MP41 and OMM2.5) cancer cells underwent cytotoxic conditions by drugs: TRIAL/APO2L, Roscovitine, and valproic acid (VAP), as well as environmental stress by heat and irradiation. Cell death and cell cycle were evaluated by FACS. β-galactosidase staining assessed senescence. cfDNA was extracted from 3 mL supernatant using QIAamp nucleic acid kit and evaluated by digital droplet PCR through hot spot mutations (Mut), and a mitochondrial gene (mt-cfDNA). Isolated cfDNA was visualized by fragment size using Bioanalyzer 2100. Results: Mut and mt-cfDNA increased during cytotoxic conditions in a dose-dependent manner. Cells under apoptosis released greater mut and mt-cfDNA compared to necrotic and untreated cells. Similarly, cell cycle disturbances by VAP were associated with an increase in Mut and mt-cfDNA. Moreover, β-galactosidase positive cells (5D post-irradiation) showed lower Mut and mt-cfDNA levels than control conditions. Electropherogram images showed that cytotoxic conditions alter fragment size distribution. While fragments about 100-200 bp were observed in apoptosis, necrosis showed more abundant fragments about >1000 bp. Conclusions: The release of cfDNA is influenced by cytotoxic and stress conditions. Knowing the biology of cfDNA can help understand its role in cancer development and how best to utilize this marker clinically, especially post cytotoxic agents that induce different cell death mechanisms. These findings have major implications in the interpretation of ctDNA in liquid biopsy as a biomarker of treatment response and tumor progression. Citation Format: Prisca Bustamante, Thupten Tsering, Julia Valdemarin Burnier. Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells [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 1694.

Characterization of cellular senescence in doxorubicin-induced aging mice.

With a rise in the need to develop anti-aging drugs, a growing number of in vivo studies evaluating the efficacy of potential drug candidates have used doxorubicin-induced aging mice. However, changes in the biomarkers of senescent cells have not been reported in detail in these animals. To lay a foundation for the use of doxorubicin-induced aging mice, we examined the biomarkers of hepatic and renal senescent cells in these mice. We found that the 5mg/kg doxorubicin dose is optimal to induce cellular senescence in mice. Subsequently, using this dose, we found that doxorubicin-induced an increase in senescence-associated β-galactosidase (SA-β-gal) positive cells in the kidney and lipofuscin accumulation in the liver. Some markers of senescent cells (p21WAF1/CIP1, p16INK4A, and γH2AX) were also significantly upregulated by doxorubicin and then counteracted by metformin treatment. These preliminary findings support the application of doxorubicin-induced aging mice as an animal model to evaluate the efficacy of anti-aging drug candidates.

Involvement of Type I Interferon Signaling in Muscle Stem Cell Proliferation During Dermatomyositis.

The idiopathic inflammatory myopathy dermatomyositis is an acquired disease that involves muscle, lung, and skin impairments. Patients with dermatomyositis show a wide range of severity of proximal skeletal muscle weakness, associated with inflammatory infiltrates, vasculitis, capillary dropout, and perifascicular myofiber atrophy. Muscles of patients with dermatomyositis show signs of muscle regeneration. Because muscle stem cells (MuSCs) are responsible for myofiber repair, we wondered whether the proliferative properties of MuSCs are altered in dermatomyositis muscle. We investigated the role of type I interferon (IFN-I) in this process because dermatomyositis is associated with sustained inflammation with high IFN-I levels. MuSCs isolated from normal muscles and those from adult and juvenile patients with dermatomyositis were grown in culture and analyzed in vitro for their proliferating properties, myogenic capacities, and senescence. Gain- and loss-of-function experiments were performed to assess the role of IFN-I signaling in the proliferative capacities of MuSCs. MuSCs derived from 8 adult patients with dermatomyositis (DM-MuSCs) (5 severe form and 3 mild form, established from histologic evaluation), from 3 patients with juvenile dermatomyositis, and from normal muscle were used to analyze their myogenesis in vitro. DM-MuSCs exhibited strongly reduced proliferating capacities as compared with healthy MuSCs (-31% to -43% for mild and severe dermatomyositis, respectively), leading to poor myotube formation (-36% to -71%). DM-MuSCs were enriched in senescent, β-galactosidase-positive cells, partly explaining the proliferation defect. Gain- and loss-of-function experiments were performed to assess the role of IFN-I on the proliferative capacity of MuSCs. High concentrations of IFN-I decreased the proliferation of healthy MuSCs. Similarly, conditioned medium from DM-MuSCs decreased the proliferation of healthy MuSCs (-15% to -22%), suggesting the delivery of an autocrine effector. Pharmacologic blockade of IFN signaling (using ruxolitinib or anti-IFN receptor antibodies) in DM-MuSCs rescued their proliferation up to the control values. These results show that autocrine IFN-I signaling prevents MuSC expansion, leading to muscle repair deficit. This process may explain the persistent muscle weakness observed in patients with severe dermatomyositis.

Nintedanib ameliorates oxidized low-density lipoprotein -induced inflammation and cellular senescence in vascular endothelial cells

ABSTRACT Atherosclerosis (AS) is a life-threatening cardiovascular disease and it has been reported that endothelial dysfunction is the initial inducer of AS. Recent reports suggest that inflammation and oxidative stress-induced cell senescence are main inducers of endothelial dysfunction. Nintedanib is an effective inhibitor of multityrosine kinase receptors developed for the treatment of fibrosis, which was recently reported to exert inhibitory effects against inflammation and oxidative stress. The present study plans to study the effect and mechanism of Nintedanib on endothelial dysfunction. We found that in oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs), the increased production of total cholesterol (TC), free cholesterol (FC), and pro-inflammatory cytokines were observed, reversed by 10 μM and 25 μM Nintedanib. The elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as the declined activity of glutathione peroxidase (GSH-Px) in ox-LDL-treated HUVECs, were significantly abolished by 10 μM and 25 μM Nintedanib. Increased proportion of senescence-associated β-galactosidase (SA-β-gal) positive staining cells, activated p53/p21 pathway, and promoted cell fraction in the G0/G1 phase were observed in ox-LDL-treated HUVECs, all of which were dramatically reversed by 10 μM and 25 μM Nintedanib. Lastly, the increased expression level of Arginase-II (Arg-II) in HUVECs by ox-LDL was repressed by Nintedanib. The protective effects of Nintedanib on ox-LDL- induced cellular senescence were pronouncedly blocked by the overexpression of Arg-II. Collectively, our data suggest that Nintedanib mitigates ox-LDL-induced inflammation and cellular senescence in vascular endothelial cells by downregulating Arg‐II.

IL-34 Downregulation‒Associated M1/M2 Macrophage Imbalance Is Related to Inflammaging in Sun-Exposed Human Skin

Macrophages can be polarized into two subsets: a proinflammatory (M1) or an anti-inflammatory (M2) phenotype. In this study, we show that an increased M1-to-M2 ratio associated with a decrease in IL-34 induces skin inflammaging. The total number of macrophages in the dermis did not change, but the number of M2 macrophages was significantly decreased. Thus, the M1-to-M2 ratio was significantly increased in sun-exposed aged skin and positively correlated with the percentage of p21+ and p16+ senescent cells in the dermis. The supernatant of M1 macrophages increased the percentages of senescence-associated β-galactosidase‒positive cells, whereas the supernatant of M2 macrophages decreased the percentages of senescence-associated β-galactosidase‒positive cells in vitro. Among the mechanisms that could explain the increase in the M1-to-M2 ratio, we found that the number of IL-34+ cells was decreased in aged skin and negatively correlated with the M1-to-M2 ratio. Furthermore, IL-34 induced the expression of CD206 and IL-10, which are M2 macrophage markers, in an in vitro assay. Our results suggest that a reduction in epidermal IL-34 in aged skin may skew the M1/M2 balance in the dermis and lead to low-grade chronic inflammation and inflammaging.

Abstract 12533: Progerin Causes a Disproportionate Senescence in Endothelial Cells

Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by the mutation of LMNA gene leading to irreversibly farnesylated lamin A protein, i.e. progerin. The major causes of death in HGPS are coronary and arterial occlusive disease. In the murine model of HGPS, vascular smooth muscle cells (VSMCs) loss is the primary vascular manifestation, which is quite different from the human arterial occlusive disease. To identify the mechanisms of HGPS vascular disease in humans, we differentiated isogenomic endothelial cells (ECs) and VSMCs from the same lines of HGPS-iPSCs and control-iPSCs. Both HGPS-ECs and HGPS-VSMCs manifested cellular hallmarks of aging, including dysmorphic nuclei, impaired proliferation, increased β-galactosidase staining, shortened telomeres, upregulated secretion of inflammatory cytokines, increased DNA damage, loss of heterochromatin and altered shelterin complex expression. However, at similar passage number, HGPS-ECs are more severely affected compared to VSMCs, as indicated by a higher percentage of β-galactosidase positive cells, significantly shortened telomere length, and greater frequency of DNA damage signals. With increasing passage number, there is progressive senescence in HGPS-ECs that is greater than that observed in HGPS-VSMCs. Using immunoprecipitation and mass spectrometry, we compared the integrity of the Shelterin Protein Complex (SPC), which is composed of six different proteins and whose function is to protect the telomeres, on the ECs and VSMCs of HGPS and control cells. We observed increased γH2A.X binding to RAP1 (member of SPC) and to Lamin A (nuclear envelope) in HGPS cells. The expression of γH2A.X was greater in HGPS ECs than VSMCs, and is associated with loss of SPC integrity, telomere shortening and loss of heterochromatin formation. Whereas progerin expression has deleterious effect in both vascular ECs and VSMCs, the severity of dysfunction is more extensive in HGPS-ECs in comparison to HGPS-VSMCs. These studies indicate that an endothelial-targeted therapy may be useful in HGPS.