DNA Damage Research Articles

METTL3 confers oxaliplatin resistance through the activation of G6PD-enhanced pentose phosphate pathway in hepatocellular carcinoma.

Oxaliplatin-based therapeutics is a widely used treatment approach for hepatocellular carcinoma (HCC) patients; however, drug resistance poses a significant clinical challenge. Epigenetic modifications have been implicated in the development of drug resistance. In our study, employing siRNA library screening, we identified that silencing the m6A writer METTL3 significantly enhanced the sensitivity to oxaliplatin in both in vivo and in vitro HCC models. Further investigations through combined RNA-seq and non-targeted metabolomics analysis revealed that silencing METTL3 impeded the pentose phosphate pathway (PPP), leading to a reduction in NADPH and nucleotide precursors. This disruption induced DNA damage, decreased DNA synthesis, and ultimately resulted in cell cycle arrest. Mechanistically, METTL3 was found to modify E3 ligase TRIM21 near the 3'UTR with N6-methyladenosine, leading to reduced RNA stability upon recognition by YTHDF2. TRIM21, in turn, facilitated the degradation of the rate-limiting enzyme of PPP, G6PD, through the ubiquitination-proteasome pathway. Importantly, high expression of METTL3 was significantly associated with adverse prognosis and oxaliplatin resistance in HCC patients. Notably, treatment with the specific METTL3 inhibitor, STM2457, significantly improved the efficacy of oxaliplatin. These findings underscore the critical role of the METTL3/TRIM21/G6PD axis in driving oxaliplatin resistance and present a promising strategy to overcome chemoresistance in HCC.

Efficacy and Tolerability of Olaparib Plus Paclitaxel in Patients with Gastric Cancer Associated with Hereditary Breast and Ovarian Cancer

Helicobacter pylori, a gram-negative, flagellated, helical bacterium, is a common cause of chronic gastric infection worldwide. According to the World Health Organization, H. pylori infection, a specific carcinogenic factor, was the leading cause of gastric cancer (GC) in 2014 worldwide (80%). H. pylori infection causes GC in >98% of patients in East Asian countries, including Japan. However, only some types of GCs are associated with H. pylori infection. Previous clinical studies have revealed that the bacterium secretes cytotoxin-associated gene A antigen, which inhibits the nuclear translocation of the breast cancer susceptibility gene 1 and 2 (BRCA1/2), a factor involved in DNA damage repair. This indicated an association between hereditary breast and ovarian cancers (HBOCs) and the development of GC. However, the detailed mechanisms underlying the development of GC caused by H. pylori infection remain unclear. Using the information on hereditary cancers obtained based on cancer genomic medicine, this study revealed that the incidence of GC was high in families with HBOC, with a preponderance for men from families with HBOC. Furthermore, the use of poly-adenosine diphosphate-ribose polymerase inhibitors in patients with hereditary GC is considered safe and effective. This study provides substantial evidence for guiding the establishment of early treatment for patients with advanced-stage/metastatic GC who harbored BRCA1/2 mutations.

Global transcriptional response of Pseudomonas aeruginosa to UVA radiation.

Ultraviolet A (UVA) radiation is the major fraction of UV radiation reaching the Earth's surface. Its harmful effects on microorganisms, due mainly to oxidative damage, have been exploited for development of natural solar and commercial UVA-based disinfection methods. In this work, the global transcriptional response of Pseudomonas aeruginosa exposed to ultraviolet A (UVA) radiation was analyzed. To conduct this study, we analyzed the whole transcriptome of the PAO1 strain grown to logarithmic phase under sublethal doses of UVA or in the dark. We found that a total of 298 genes responded to UVA with a change of at least two-fold (5.36% of the total P. aeruginosa genome), and showed equal amount of induced and repressed genes. An important fraction of the induced genes were involved in the response to DNA damage and included induction of SOS, prophage and pyocins genes. The results presented in this study suggest that one of the main UVA targets are proteins carrying [Fe-S] clusters since several genes involved in the processes of synthesis, trafficking and assembly of these structures were upregulated. The management of intracellular iron levels also seems to be a robust response to this stress factor. The strong induction of genes involved in denitrification suggest that this pathway and/or reactive nitrogen species such as nitric oxide could have a role in the response to this radiation. Regarding the down-regulated genes, we found many involved in the biosynthesis of PQS, a quorum-sensing signal molecule with a possible role as endogenous photosensitizer.

The kinase ATR controls meiotic crossover distribution at the genome scale in Arabidopsis

Abstract Meiotic crossover, i.e., the reciprocal exchange of chromosome fragments during meiosis, is a key driver of genetic diversity. Crossover is initiated by the formation of programmed DNA double-strand breaks (DSBs). While the role of ATAXIA-TELANGIECTASIA AND RAD3-RELATED (ATR) kinase in DNA damage signaling is well-known, its impact on crossover formation remains understudied. Here, using measurements of recombination at chromosomal intervals and genome-wide crossover mapping, we showed that ATR inactivation in Arabidopsis (Arabidopsis thaliana) leads to dramatic crossover redistribution, with an increase in crossover frequency in chromosome arms and a decrease in pericentromeres. These global changes in crossover placement were not caused by alterations in DSB numbers, which we demonstrated by analyzing phosphorylated H2A.X foci in zygonema. Using the seed-typing technique, we found that hotspot usage remains mainly unchanged in atr mutants compared to wild-type individuals. Moreover, atr showed no change in the number of crossovers caused by two independent pathways, which implies no effect on crossover pathway choice. Analyses of genetic interaction indicate that while the effects of atr are independent of MMS AND UV SENSITIVE81 (MUS81), ZIPPER1 (ZYP1), FANCONI ANEMIA COMPLEMENTATION GROUP M (FANCM) and D2 (FANCD2), the underlying mechanism may be similar between ATR and FANCD2. This study extends our understanding of ATR’s role in meiosis, uncovering functions in regulating crossover distribution.

Extensive DNA Damage and Loss of Cell Viability Occur Synergistically With the Combination of Recombinant Methioninase and Paclitaxel on Pancreatic Cancer Cells which Report DNA-Damage Response in Real Time.

Methionine restriction selectively arrests cancer cells during the S-phase of the cell cycle. We hypothesized that DNA damage may occur in S-phase in cancer cells during methionine restriction. To determine if this occurs, we used MiaPaCa-2Tet-On 53BP1-green fluorescent protein (GFP) pancreatic cancer cells, which report GFP fluorescence in real time after DNA-damage response (DDR) in these cells. We also determined whether a chemotherapy drug in combination with methionine restriction increases the rate of DNA damage. MiaPaCa-2Tet-On 53BP1-GFP cells were used for in vitro experiments. The 25% and 50% inhibitory concentrations (IC25 and IC50, respectively) of recombinant methioninase (rMETase) and paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP pancreatic cancer cells were determined. Cell viability and DDR with rMETase alone, paclitaxel alone, and their combination were measured in MiaPaCa-2Tet-On 53BP1-GFP cells. The IC25 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 1.66 U/ml. The IC25 for paclitaxel on MiaPaCa-2Tet-On 53BP1-GFP cells was 3.31 nM. The combination of rMETase and paclitaxel synergistically reduced the viability of MiaPaCa-2Tet-On 53BP1-GFP cells. The IC50 of paclitacel on MiaPaCa-2Tet-On 53BP1-GFP cells was 5.1 nM. The IC50 of rMETase on MiaPaCa-2Tet-On 53BP1-GFP cells was 2.3 U/ml. The combination of rMETase (IC50) plus paclitaxel (IC50) on MiaPaCa-2Tet-On 53BP1-GFP cells also caused more DNA damage than either agent alone. The present study suggests the synergy of methionine restriction and chemotherapy is due, at least in part, to DNA damage of cancer cells.

Erp57 facilitates ZIKV-induced DNA damage via NS2B/NS3 complex formation.

It is believed that DNA double-strand breaks induced by Zika virus (ZIKV) infection in pregnant women is a main reason of brain damage (e.g. microcephaly, severe brain malformation, and neuropathy) in newborn babies [1,2], but its underlying mechanism is poorly understood. In this study, we report that the depletion of ERp57, a member of the protein disulphide isomerase (PDI) family, leads to the limited production of ZIKV in nerve cells. ERp57 knockout not only suppresses viral induced reactive oxygen species (ROS) mediated host DNA damage, but also decreases apoptosis. Strikingly, DNA damage depends on ERp57-bridged complex formation of viral protein NS2B/NS3. LOC14, an ERp57 inhibitor, restricts ZIKV infection and virus-induced DNA damage. Our work reveals an important role of ERp57 in both ZIKV propagation and virus-induced DNA damage, suggesting a potential target against ZIKV infection.

Myocardial DNA damage is responsible for the relationship between genotype and treatment response in patients with dilated cardiomyopathy

Abstract Background Dilated cardiomyopathy (DCM) is one of the leading causes of heart transplantation. Our group and others have previously reported that DNA damage accumulation in myocardial tissue and certain genetic variants such as those in LMNA are associated with treatment response and prognosis (ref 1-5). However, the potential association between myocardial DNA damage and genotype, and their contribution when combined to predicting treatment response, remains unclear. Methods We identified the deleterious variants in cardiomyopathy-related genes by analyzing the whole exome sequencing data from 89 patients with DCM who were recruited from two participating facilities. Immunostaining of γ-H2A.X, a DNA damage marker, was performed on endomyocardial biopsy specimens from the same patients, from which the percentage of nuclei positive for γ-H2A.X signals (%γ-H2A.X) was calculated. Left ventricular reverse remodeling (LVRR) was assessed by transthoracic echocardiography at 1 year after biopsy. We examined the associations among genotype, myocardial DNA damage, and the achievement of LVRR. Results Among the 89 patients with DCM, 11 (12.4%) carried pathogenic or likely pathogenic (P/LP) variants in sarcomere genes such as TTN, and 21 (23.6%) carried P/LP variants in non-sarcomere genes such as LMNA, while the rest had no P/LP variants in cardiomyopathy-related genes (Figure A). Patients with P/LP variants in sarcomere genes had a lower %γ-H2A.X [21.2 (10.7–29.0)% vs. 7.0 (4.3–12.4)%, P = 0.002] (Figure B), and a higher probability to achieve LVRR at 1-year (81.8% vs. 23.8%, P = 0.003) (Figure C) when compared to those with P/LP variants in non-sarcomere genes. The odds ratio for carriers of P/LP variants in sarcomere genes to achieve LVRR was 14.40 (95%CI 2.31–89.94) compared to that for carriers of P/LP variants in non-sarcomere genes. However, this effect was markedly attenuated after adjusting for the proportion of γ-H2A.X-positive nuclei [adjusted odds ratio 6.05 (95%CI 0.86–42.53)] (Table). Conclusions Among patients with DCM, carriers of P/LP variants in sarcomere genes showed less DNA damage and a favorable treatment response, when compared with carriers of P/LP variants in non-sarcomere genes. Our results suggest that myocardial DNA damage was partially responsible for the relationship between genotype and treatment response in patients with DCM.Figure, panels A-C

Electronegative ApoCIII-Rich HDL promotes cellular damage and vascular aging

Abstract Background Experimental studies attributing HDL a cardioprotective effect are challenged by well-designed randomized controlled trials showing largely neutral results, which might be related to more recent observations that certain HDL particles exert detrimental effects. This study explores the effects of electronegative HDL on cellular damage and vascular aging. Using anion-exchange chromatography, HDL was divided into five subclasses (H1 to H5) based on increasing electronegativity. Compared to the health-promoting H1, the most electronegative subclass, H5 contains significantly higher levels of ApoCIII and triglycerides (TG). Purpose Herein, we aim to explore the cellular and molecular mechanisms underpinning H5-induced pathology in human umbilical vein vascular endothelial cells (HUVECs), which may accelerate vascular aging preceding the development of atherosclerotic cardiovascular disease (ASCVD). Methods H1 and H5 were isolated from patients with metabolic syndrome (MetS). To gauge the impact of H5 on vascular integrity, HUVECs were treated with solvent, H1, or H5 at concentrations of 25, 50, or 100 µg/mL. Cell viability and apoptosis were evaluated using the MTT assay and PI/Calcein fluorescent staining, respectively. Immunofluorescence staining and immunoblotting, focusing on 8-oxodG, P53, and β-gal, proxies of DNA damage and cellular senescence, were performed. Senescence-associated secretory phenotype (SASP) analysis was done to elucidate the senescent mechanism. By harnessing Mitosox imaging reactive oxygen species (ROS) synthesis was monitored. Finally, unbiased lipid- and transcriptomics were used to identify lipid components potentially involved in H5 signaling. Results As compared to controls, MetS patients exhibited more electronegative HDL particles, as indicated by increased H5 (1.604±0.410% vs 4.598±4.712%, P=0.0016). Remarkable elevations were noted in individuals with severe clinical complications, as shown in the representative patient with left ventricular hypertrophy, chronic coronary syndrome, and heart failure (Fig. 1). In HUVECs, H5 but not H1 reduced cell viability and increased apoptosis in a concentration-dependent manner. In parallel, H5 but not H1 induced cell aging, ROS production, and DNA damage, with SASP cytokines including IL-1β, IL-6, CCL2, TNF-α, and γH2AX (Fig. 1). Notably, compared to H1, H5 exhibited high contents of various TG species, impairing H5’s anti-inflammatory and antioxidant capacities, along with reductions in several phosphatidylcholines that may constitute essential cell membrane structural components, as evidenced by lipidomic studies (Fig. 2). Conclusion H5, the most electronegative and dysfunctional HDL subfraction, is capable of inducing DNA damage, oxidative stress, and senescence in vascular ECs. Its notable elevation in patients with MetS highlights a previously unrecognized etiology, shedding light on how electronegative HDL may contribute to and expedite ASCVD.Fig 1Fig 2

Intact DNA repair in differentiated cardiomyocytes is essential for maintaining cardiac function in response to pressure overload in mice

Abstract Introduction DNA in every cell is continuously damaged and DNA repair mechanisms are essential for protection against DNA damage-induced aging-related diseases. For example, deficient repair of endogenously generated DNA damage in mice with cardiomyocyte-restricted inactivation of Xpg, is associated with progressive heart failure. Purpose Here we tested the hypothesis that unrepaired DNA damage in differentiated cardiomyocytes increases cardiac vulnerability in response to hemodynamic overload. Methods To increase the burden of spontaneous DNA damage, we generated mice with cardiomyocyte-restricted inactivation of DNA repair endonuclease XPG. At 8 weeks of age, αMHC-Xpgc/- and control (Ctrl) mice were subjected to pressure overload by transverse aortic constriction (TAC). Eight weeks after TAC, left ventricular (LV) function was assessed using echocardiography and hemodynamic measurements, followed by molecular and histological analyses. Results Cardiomyocyte-restricted inactivation of Xpg resulted in systolic as well as diastolic LV dysfunction (Table). TAC-induced LV hypertrophy is similar in both groups (Ctrl 38%; αMHC-Xpgc/- 34%). In Ctrl mice, LV hypertrophy was accompanied by minimal LV dilation and modest changes in systolic and diastolic LV function. Conversely, TAC in αMHC-Xpgc/- produced severe LV dysfunction and resulted in overt congestive heart failure, demonstrated by aggravation of LV dilation, and marked increases in LV end-diastolic pressure, left atrial weight and lung fluid weight, which was accompanied by further increases in the expression levels of the hypertrophic marker genes atrial natriuretic peptide and beta-myosin heavy chain (Table). Moreover, lectin staining revealed a decrease in capillary density and TUNEL staining revealed further elevated levels of myocardial apoptosis. In addition, a significant increase of myocardial collagen content was observed. Conclusion Cardiomyocyte-restricted loss of DNA repair protein XPG increases cardiac vulnerability to develop heart failure in response to pressure overload. These findings underscore the importance of genomic stability for maintenance of cardiac function, not only during basal conditions, but also in response to a pathological stimulus.

Mature cardiac tissues modeling Duchenne muscular dystrophy related cardiomyopathy

Abstract Background The major cause of mortality in patients with Duchenne muscular dystrophy (DMD) is cardiomyopathy. This disease is caused by dystrophin gene mutations that result in the loss of dystrophin protein. Gene replacement therapies have been approved for skeletal muscle symptoms of DMD. However, these therapeutic modalities are not indicated for cardiomyopathy due to their low efficiency of delivery to the heart. Despite the significant unmet needs, a limitation in this area of research is the divergence of cardiomyopathy symptoms in model animals from those in patients. Alternatively, cardiomyocytes differentiated from patient-derived iPS cells have recently been analyzed. However, existing results are based on a single time point analysis and do not model disease progression or non-cardiomyocyte involvement. Purpose Our research aims to establish an in vitro model that can reproduce the pathological progression of DMD cardiomyopathy. Methods We have generated cardiac organoids and engineered heart tissues (EHTs) from patient-derived iPS cells, consisting of cardiomyocytes and epicardial-derived non-cardiomyocytes. An iPS cell line in which the dystrophin mutation was repaired by genome editing was used as a control. The cardiac organoids or EHTs were subjected to transient activation of fatty acid metabolism to promote the maturation of iPS-derived cardiomyocytes. Pathological conditions were then induced in these 3D-models by applying loads that mimicked the DMD-disease environment. Results The mature cardiac organoids displayed adult-like gene expression profiles in the components of dystrophin-associated-protein-complex and calcium handling. After continuous mechanical loading, cell death appeared in DMD-EHTs, although there was only a slight decrease in contractility and fibrosis. Elevated mitochondrial ROS production and DNA damage were observed as a part of the cell death mechanism. Furthermore, pathological stimuli in addition to mechanical loading reproduced progressive contractile dysfunction and further increase in fibrosis in DMD-EHTs. Conclusions From these observations, we concluded that our DMD-EHT model recapitulate the pathological development of DMD-cardiomyopathy. These results suggest that the key factors involved in the progression of DMD cardiomyopathy are (i) the fragility of cardiomyocytes to stress in early stages and (ii) the involvement of non-cardiomyocytes such as fibrosis. Our model provides insight into the molecular mechanisms underlying disease progression and novel therapeutic targets.

STING-mediated DNA damage response in diabetes-induced vascular complications

Abstract Background Hyperglycemia drives endothelial dysfunction, a key factor in the development of diabetes vascular complications. The stimulator of interferon genes (STING) signal, plays a crucial role in the immune system and is implicated in the pathogenesis of inflammatory diseases. Therefore we investigated the role of STING in endothelial dysfunction in streptozotocin (STZ)-induced diabetic mice. Methods and Results Diabetes was induced by single-dose administration of STZ in 8-week-old C57BL/6 and Sting-/- mice. STZ injection promoted the expression of STING and DNA damage markers in the aorta of C57BL/6 mice. Genetic deletion of STING ameliorated endothelial dysfunction as determined by vascular reactivity assay (P < 0.001) and increased aortic eNOS phosphorylation (P < 0.05) in STZ-injected mice. Stimulation of STING agonist (cGAMP), or mtDNA increased inflammatory molecules expression (e.g., VCAM1 and IFNB) and decreased eNOS phosphorylation in HUVECs. In ex-vivo experiments, cGAMP significantly impaired endothelial function which was ameliorated in the presence of an STING inhibitor or a neutralizing IFN-β antibody. Furthermore, the administration of STING inhibitor ameliorated endothelial dysfunction in STZ-induced diabetic mice (P < 0.01). Conclusion The DNA damage response regulated by STING aggravated diabetes-induced endothelial dysfunction. STING signaling may be a potential therapeutic target for diabetic vascular complications.

Long non-coding RNA PANDA promotes endothelial senescence and oxidative damage through the interaction with NRF2

Abstract Background Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. LncRNAs are emerging as important players in the pathogenesis of cardiovascular disease. PANDA, a newly identified lncRNA, is a key regulator of cellular senescence, apoptosis and oxidative stress. Purpose To investigate the role and molecular mechanism of PANDA in diabetic vascular disease. Methods RNAseq was performed to aorta specimen from diabetic and no diabetic patients as well to HAECs exposed to 5 mM and 25 mM concentrations (Normal -NG- and High -HG-, respectively). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled siRNA wass used as a negative control. RNAseq and network perturbation analysis (NPA) of treated HAECs unveiled transcriptional changes upon PANDA depletion. Expression of PANDA was assessed by real time PCR. PANDA RNA-IP was performed to check its binding to relevant transcriptional factor (such as NRF2) as well Ch-IP was performed to check the NRF2 binding on oxidative gene promoters. Cellular localization of NRF2 was investigated by Immunofluorescence. Beta-galactosidase and superoxide staining was used to detect endothelial senescence and ROS formation; while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. Results PANDA expression was significantly increased in diabetic human aorta as well in HG-treated HAECs (Figure1 A-B). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG cells (Figure1 C-D). Western blot analysis reveals the restored level of HMOX1 and TFRC1 upon PANDA depletion (Figure1 E-G). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. In HG, NFR2 is downregulated while PANDA silencing restores its level (Figure1 I-J). We found that in HG condition PANDA binds the transcription factor NRF2 and blocks its nuclear translocation thus impeding its binding on HMOX1 and TFRC promoters (Figure1 K-M). Silencing of PANDA in HG-treated HAECs reduced genes and cellular features of senescence (Figure2 A-B), restored the expression of anti-apoptotic genes and decreases caspase 3 activity (Figure2 C-D), improved endothelial migration and tube formation (Figure2 E and G), and reduced ROS formation (Figure2 F). Conclusions In hyperglycemia PANDA upregulation drives endothelial senescence while impairing angiogenic properties. On the other hand, PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes through the release of NRF2 that in turn translocate into the nucleus enhancing the expression of the antioxidant gene HMOX1. The results indicate PANDA as a putative novel molecular target in the setting of diabetic vascular disease (Figure2 H).

Modulation of p300 acetylation to protect against doxorubicin-induced cardiotoxicity

Abstract Background Doxorubicin (DOX) is a widely-used anti-neoplastic agent, but the most common adverse reaction to its use is cardiotoxicity(1). DOX acts by producing reactive oxygen species (ROSs) that cause DNA damage(1,2), which activates various proteins, including the tumour suppressor p53 and the acetyltransferase p300. When activated, p300 acetylates and increases the activity of p53 which leads to increased apoptotic activity(3). Cardiotoxicity results because the heart has limited mechanisms to dispose of ROSs(4), and because cardiomyocytes have a low turnover rate(5). Purpose We examined the p300 inhibitor Theracurmin (THR)(6,7) as a candidate to prevent DOX cardiotoxicity using an in-vivo mouse model, and sought to elucidate the underlying molecular mechanisms using in-vitro studies. Methods C57BL/6 mice were pre-treated with THR or vehicle (as control), then administered DOX or vehicle. Cardiac function was evaluated by echocardiography and cardiac catheterization. In vitro studies used cardiac fibroblasts (FBs) and cardiac endothelial cells (ECs) pre-treated with THR, then with DOX, as in the animal studies. Western blotting and rt-qPCR was used to quantify protein and mRNA levels of genes in the p53-p300 pathway or related to apoptosis, oxidative stress, and cell cycle control. Results DOX-treated mice showed significant decreases in left ventricular (LV) ejection fraction (LVEF, p=0.0004), fractional shortening (FS, p=0.0012), and a significant increase in end-systolic volume (ESV, p=0.0004). We also showed a significant decrease in anterior (LVAW;d, p=0.005) and posterior (LVPW;d, p=0.005) wall thickness of the LV in these mice. However, when mice were pre-treated with THR prior to DOX administration, we saw significant increases in LVEF (p=0.045) and LVPW;d (p=0.015), and a significant decrease in ESV (p=0.0047) compared to those that were not pre-treated. In-vitro analyses demonstrated a significant increase in cleaved caspase 3 protein expression in DOX-treated ECs (p=0.007), that was not prevented by THR pre-treatment. In DOX-treated FBs, we saw a reduction in AKT mRNA (p=0.017) and increases in BAX 9p=0.008), P21 (p=0.0001), and GADD45 (0.037) mRNA expression. In DOX-treated ECs, we also saw significant increases in BAX (p=0.015) and P21 (p=0.011) mRNA expression. These changes in mRNA expression were not reversed with THR pre-treatment in either cell type. Conclusion THR pre-treatment successfully mitigated functional deficits and structural deficits associated with DOX cardiotoxicity. In-vitro studies show that THR pre-treatment was not sufficient to significantly prevent changes in key molecular targets associated with apoptosis. Further studies are required to elucidate the mechanism behind the observed functional changes.Abstract OverviewAbstract Results

ATM: a protein involved in glucose and lipid metabolism in the heart

Abstract Backround Post-mitotic cells, such as neurons and cardiomyocytes, cannot repair DNA lesions with DNA replication and rely for their survival on efficient sensors and effectors that orchestrate the DNA damage response (DDR). The Ataxia Telangiectasia Mutated (ATM) protein kinase is the most important sensor of oxidative stress and the DNA damage response (DDR) and is implicated in cellular metabolism. It is believed that while transient DNA damage and DDR can temporarily improve cardiovascular function, persistent activation of DDR (and ATM) might promote the onset and development of heart failure (HF). Purpose We hypothesised that ATM might control and regulate energy metabolism in the heart under stress to promote DNA repair. Methods We analyzed the effects of ATM inactivation on cardiomyocyte hypertrophy, cardiac function, DDR and metabolism in hearts from wild-type (Atm+/+) or Atm-mutated (Atm-/-) mice under sham conditions or after pressure overload by transverse aortic constriction (TAC). Results ATM inactivation in Atm-/- mice induced cardiomyocyte hypertrophy, fetal gene expression re-activation and a specific metabolomic signature in the heart, characterized by significant accumulation of pyruvate, branched chain amino-acids, short-medium acyl-carnitines and metabolites of tricarboxylic acid cycle. The levels of the glycolytic enzymes, hexokinase-2 (HK2) and phosphofructokinase (PFK), were elevated. pyruvate was trapped in the cytosol because mitochondrial carriers were suppressed and the enzymes that process pyruvate were dysregulated. Because of pyruvate metabolic block, fatty acids oxidation was inefficient and resulted in the accumulation of acyl-carnitines and insulin resistance. These metabolic changes were amplified by TAC, which rapidly induced heart failure in Atm-/- mice. ATM inactivation also increased basal and TAC-induced genomic stress in cardiomyocytes, as shown by the levels of p-γ-H2AX, 8-oxodG glycosylase (OGG1/2) and apurinic site nuclease (APE1). These results prove that ATM rewires the metabolism of cardiac cells by inducing glycolysis and fatty acids oxidation. ATM stimulates glycolysis to repair DNA lesions and protect the heart against stress-induced dysfunction. The metabolic block due to ATM inactivation accelerates heart failure. Conclusions Our data suggest that ATM favors the metabolic flexibility of the heart by stimulating glucose entry and balancing glucose catabolism with fatty acid oxidation, thus preventing mechanical stress-induced cardiac systolic dysfunction.

STING-dependent induction of neutrophilic asthma exacerbation in response to house dust mite.

Severe refractory, neutrophilic asthma remains an unsolved clinical problem. STING agonists induce a neutrophilic response in the airways, suggesting that STING activation may contribute to the triggering of neutrophilic exacerbations. We aim to determine whether STING-induced neutrophilic lung inflammation mimics severe asthma. We developed new models of neutrophilic lung inflammation induced by house dust mite (HDM) plus STING agonists diamidobenzimidazole (diABZI) or cGAMP in wild-type, and conditional-STING-deficient mice. We measured DNA damage, cell death, NETs, cGAS/STING pathway activation by immunoblots, N1/N2 balance by flow cytometry, lung function by plethysmography, and Th1/Th2 cytokines by multiplex. We evaluated diABZI effects on human airway epithelial cells from healthy or patients with asthma, and validated the results by transcriptomic analyses of rhinovirus infected healthy controls vs patients with asthma. DiABZI administration during HDM challenge increased airway hyperresponsiveness, neutrophil recruitment with prominent NOS2+ARG1- type 1 neutrophils, protein extravasation, cell death by PANoptosis, NETs formation, extracellular dsDNA release, DNA sensors activation, IFNγ, IL-6 and CXCL10 release. Functionally, STING agonists exacerbated airway hyperresponsiveness. DiABZI caused DNA and epithelial barrier damage, STING pathway activation in human airway epithelial cells exposed to HDM, in line with DNA-sensing and PANoptosis pathways upregulation and tight-junction downregulation induced by rhinovirus challenge in patients with asthma. Our study identifies that triggering STING in the context of asthma induces cell death by PANoptosis, fueling the flame of inflammation through a mixed Th1/Th2 immune response recapitulating the features of severe asthma with a prognostic signature of type 1 neutrophils.

The mechanism of paclitaxel induced damage on placental trophoblast cells

ObjectiveChemotherapy during pregnancy has a certain risk of causing a series of complications, such as miscarriage, premature birth, or fetal growth restriction, although the relationship between these complications and chemotherapy is currently unclear. This experiment focuses on the possible damage mechanism of the chemotherapeutic drug paclitaxel on placental trophoblast cells, and explores whether chemotherapy can affect pregnancy outcomes by directly damaging placental tissue.MethodsThis study explored the mechanism of paclitaxel induced damage on placental trophoblast cell lines JEG-3 and BEWO through immunofluorescence staining, Western blot experiments, cell flow cytometry, Seahorese cell metabolism experiments, and mouse modeling verification.ResultsThe experiment found that paclitaxel could induce JEG-3 and BEWO cells to produce reactive oxygen species (ROS), and elevate the ratio of Bax/Bcl-2 expression. Besides, paclitaxel mediated the reduction of mitochondrial membrane potential in JEG-3 and BEWO cells, causing damage and leading to mitochondrial autophagy and the occurrence of unfolded protein response. Paclitaxel inhibited the glycolysis rate of JEG-3 and BEWO cells, and leaded to impaired mitochondrial function, including decreased basal respiratory values, decreased respiratory reserve capacity, and proton leakage. In pregnant mice with tumor modeling, paclitaxel could cause DNA damage in placental tissue cells, and might lead to apoptosis of chemotherapy mice placental tissue cells and impairment of normal physiological functions.ConclusionPaclitaxel may directly or indirectly affect the normal physiological functions of placental trophoblast cells, including energy metabolism and protein synthesis dysfunction, which may be related to the adverse pregnancy outcomes caused by paclitaxel chemotherapy.

Genetically Engineered Clostridium Prevents Perivascular Adipose Tissue and Endothelial Senescence and Dysfunction in Aging

Abstract Introduction (Peri)vascular senescence is a bona fide risk for several aging-associated diseases that threatens healthspan. Gut microbiome may be one of triggers that promotes vascular diseases as host ages. However, the interplay between vascular senescence and gut microbiome is largely unknown. Our recent studies revealed an increase in gut-derived phenylacetic acid (PAA) in the aging population (TwinsUK Aging Cohort, n=7,303) and aged mice. Our preliminary shotgun metagenomics also showed that ppfor-harboring Clostridium sp. ASF356 age-dependently generates PAA, which induces perivascular adipose tissue (PVAT) and endothelial cell (EC) senescence and dysfunction in old mice. Yet, it remains unclear whether and how genetic engineering of Clostridium sp. ASF356 (by depleting ppfor gene) can prevent aortic PVAT-EC senescence and restore vascular function in aging. Methods For bacterial genetic engineering, we depleted ppfor gene in Clostridium ASF356 by ClosTron technique and further mono-colonized aged mice (24-months old) and germ-free mice, as control, to reduce plasma PAA and thereby prevent aortic PVAT-EC senescence and improve endothelial dysfunction. Results As a proof of the concept, our studies exhibited that mono-colonization of germ-free mice with wild-type Clostridium sp. ASF356 markedly elevates plasma PAA levels. It was accompanied by hallmarks of aortic PVAT-EC senescence, including increased DNA damage (↑ γ-H2A.X phosphorylation), heightened SASP (↑ IL-6 and VCAM1), and proliferative arrest (↑ p16INK4a and p21WAF1/Cip1). Additionally, our findings revealed upreglation of Notch1 in PVAT, contributing to reduced energy supply (↓ UCP1) and augmented senescence-messaging secretome (↑ IL-6) towards ECs. Our further studies revealed that mono-colonization of aged mice (pre-treated with antibiotics) with Δppfor Clostridium ASF356 mutants intriguingly resulted in decreased plasma PAA levels. This intervention led to the rescues of cellular senescence in both aortic PVAT and ECs, consequently improving vascular function. Specifically, mice subjected to this treatment exhibited a significant reduction in SA-β-galactosidase+ cells, as well as decreased levels of γ-H2A.X phosphorylation, p16INK4a, p21WAF1/Cip1, and IL-6 in their aortic PVAT and ECs compared to wild-type aged mice. Conclusions The current study suggests that genetically engineered Δppfor Clostridium ASF356 mutants reverse cellular senescence and restore function in aortic PVAT and endothelial cells, highlighting the potential for targeted gut microbiome manipulation as an innovative senotherapy to combat vascular aging.

Biological effects of radiation exposure in patients treated with X-ray guided endovascular aortic repair

Abstract Background X-ray guided cardiovascular interventions such as endovascular aortic repair (EVAR) expose patients and operators to significant amounts of repeated ionising radiation over a long period of time. The biological effect of such protracted radiation exposure is poorly understood. We have previously demonstrated a higher incidence of dicentric chromosome (DC) aberrations in high-volume endovascular interventionalists and differences in acute DNA damage in lymphocytes between individuals following in vitro irradiation. Studies suggested a higher incidence of cancer in patients who underwent EVAR compared with those who have had open aneurysm repair, but the evidence is inconclusive. Further work is required to evaluate the biological radiation risk of X-ray guided cardiovascular interventions. Purposes We aimed to examine radiation-induced genome instability from patients after complex EVAR. We also aimed to investigate the intrinsic response of each patient to radiation using biomarkers of acute DNA damage following in vitro irradiation of their blood and to correlate this response to the expression of radiation-responsive genes. Methods Lymphocytes were isolated from patients after complex (branched/fenestrated) EVAR and non-irradiated controls. DC, a type of chromosomal aberration caused by radiation exposure were enumerated. γ-H2AX, a marker of acute DNA damage/repair, was measured by immunofluorescence after in vitro irradiation at 0.2 Gy and 1 Gy, along with the expression of the radiation-responsive genes FDXR, CCNG1, P21 and PHPT1 using qPCR. Results 17 patients (82% male, median age 73[59 – 85years]) and 16 controls (56% male, median age 68[53 – 83years]) were recruited. The mean incidence of DC was 3.782 (95% CI 3.13 - 4.43) and 0.898 (95% CI 0.48 -1.32) per 1000 cells for patients and controls, respectively (P<0.0001). Patients had higher background of γ-H2AX foci than unexposed controls, (0.7056, 95% CI 0.316 - 1.10) vs (0.241 95% CI 0.077 - 0.405) per cell, P<0.05. FDXR expression was most increased following irradiation (P<0.0001), reaching a mean 13.1-fold and 11.4-fold increase at 4-hour and 24-hour time-point, respectively, but this upregulation was not significantly different between the two groups. Conclusion We have shown an increased frequency of chromosomal aberrations in patients after complex EVAR, a biological finding that may support a propensity to developing malignancies. Patients also have higher basal DNA damage/repair (γ-H2AX) activity. Expression of ferredoxin reductase, FDXR gene could be a potential radio-responsive biomarker to provide personalised biological dosimetry information for clinical monitoring. Our study further underlines the long-term radiation risk of X-ray guided cardiovascular interventions and the potential of biological assays that may guide personalised care.

Elevated α/β ratio after hypofractionated radiotherapy correlated with DNA damage repairment in an experimental model of prostate cancer.

Our previous study demonstrated that the linear quadratic model appeared to be not well-suited for high dose per fraction due to an observed increase in α/β ratio as the dose per fraction increased. To further validate this conclusion, we draw the cell survival curve to calculate the α/β ratio by the clone formation experiment and then convert the fractionated radiation dose into an equivalent single hypofractionated radiation dose comparing with that on the survival curve. Western Blot and laser confocal immunofluorescence were used to detect the expression of γ-H2AX and RAD51 after different fractionated modes of radiation. We constructed a murine xenograft model, and changes in transplanted tumor volume were used to evaluate the biological effects after different fractionated radiation. The results demonstrated that when fractionated radiation dose was converted into equivalent single hypofractionated radiation dose, the effectiveness of hypofractionated radiation was overestimated. If a larger α/β ratio was used, the discrepancy tended to become smaller. γ-H2AX was higher in 24h after a single high dose radiation than the continuous expression of the DNA repair marker RAD51. This implies more irreparable damage in a single high dose radiation compared with fractionated radiation. In the murine xenograft model, the effectiveness of hypofractionated radiation was also overestimated, and additional fractions of irradiation may be required. The conclusion is that after single hypofractionated radiation, the irreparable damage in cells increased (α value increased) and some repairable sublethal damage (β value) was converted into irreparable damage (α value). When α value increased and β value decreased, the ratio increased.

Activation, regulation, and effects of the mitochondrial unfolded protein response in endothelial cells

Abstract Introduction Endoplasmic reticulum stress and the resulting unfolded protein response (UPRER) play significant roles in endothelial dysfunction and atherosclerosis. However, the understanding of the mitochondrial unfolded protein response (UPRMito) in endothelial dysfunction is limited. This study aims to characterize the activation, regulation, and effects of the UPRMito to provide a comprehensive understanding of endothelial UPRMito. Methods and Results RNA sequencing and quantitative proteomic analysis were employed to investigate the impact of stressors on mitochondrial (MitoBlock-6, CDDO) and endoplasmic (Tunicamycin) protein homeostasis on the transcriptome and proteome of Human Coronary Artery Endothelial cells (HCAEC, Fig.1). The analysis identified 12,745 unique transcripts and 7,891 unique proteins. Mitocarta annotation highlighted the regulatory effect induced by these stressors, particularly on mitochondrial proteins and transcripts (Fig. 2). Geneset enrichment analyses revealed upregulation of pathways regulated by activating transcription factors 4/5 (ATF4/5) and the integrated stress response marker CHOP. Analysis of protein expression changes unveiled that 21 proteins were upregulated by both CDDO and MitoBlock-6, including essential mitochondrial chaperones (HSP10 and HSP60). In contrast, the UPRER stressor Tunicamycin did not induce canonical UPRMito activation. Additionally, 142 proteins were commonly downregulated, affecting crucial biological processes such as mitochondrial translation, mitochondrial gene expression, and ribosome biogenesis. Importantly, NAD+ADP-ribosyltransferase activity was highly downregulated, involving PARP proteins with roles in DNA damage repair. On a functional level, mitochondrial stressors led to dose-dependent alterations in HCAEC viability, apoptosis, and reactive oxygen species formation (ROS) measured by resazurin-based assays, caspase 3/7 activity, and MitoSox staining, respectively. Low doses and short incubations with mitochondrial stressors promoted endothelial cell health through cytoprotective UPRMito pathways, while prolonged incubation led to unresolvable mitochondrial stress (exemplarily shown in Fig. 3). SiRNA-mediated gene silencing of the UPRMito regulators ATF4 and ATF5 promoted mitochondrial ROS formation in cells treated with mitochondrial stressors. These effects were particularly more pronounced after ATF5-Knockdown, suggesting that ATF5 is the main regulator of the UPRMito in HCAEC (Fig. 4). Moreover, high-dose UPRMito stressors led to an impairment in mitochondrial membrane integrity with cytosolic release of pro-inflammatory mtDNA. Conclusion UPRMito and UPRER lead to a different stress response. Depending on the underlying conditions, both stress responses can exert both cytoprotective and cytotoxic effects. Further studies are required to analyze the in vivo relevance and possible therapeutic exploitation of the UPRMito in endothelial dysfunction and atherosclerosis.Figures