Cross-sectional and longitudinally, no renal outcome was associated with PM2.5 or BC in models adjusted for sex and baseline or time varying covariables, including age, blood pressure, heart rate, body mass index, plasma glucose, the total-to-HDL serum cholesterol ratio, alcohol intake, smoking, physical activity, socioeconomic class, and antihypertensive treatment. The subject-level geocorrelations of eGFR change with to BC and PM2.5 were 0.13 and 0.02, respectively (P ≥ 0.68). In conclusion, in a population with moderate exposure, renal function was unrelated to ultrafine particulate.From 1990 until 2017, global air-pollution related mortality increased by 40%. Few studies addressed the renal responses to ultrafine particulate [≤ 2.5 µm (PM2.5)], including black carbon (BC), which penetrate into the blood stream. In a Flemish population study, glomerular filtration estimated from serum creatinine (eGFR) and the urinary albumin-to-creatinine ratio were measured in 2005–2009 in 820 participants  with follow-up of 523 after 4.7 years (median). Serum creatinine, eGFR, chronic kidney disease (eGFR < 60 mL/min/1.73 m The subsequent 2017 report confirmed this trend and highlighted the contribution of environmental pollutants, including air pollution, as driver of chronic kidney disease (CKD)2. From 1990 until 2017, global mortality and disability adjusted life-years (DALYs) related to ambient air pollution by particulate matter increased by 39.35% and 43.45%, respectively2.According to the report of the Global Burden of Disease Study, renal failure contributed to 0.403 million death in 1990 which increased to 0.736 million in 2010 worldwide2.5) reaches the smallest airways and alveoli, crosses the blood-air barrier and directly penetrates into the blood stream3. Black carbon (BC), a component of PM2.5, consists of pure carbon in several bond forms, and finds its origin in the incomplete combustion of fossil fuels, biofuel or biomass. The kidneys clear BC particles from the circulating blood4. The number of carbon particles in urine is measurable and reflects chronic exposure to combustion-related air pollution4. Several studies reported on the levels of particulate matters in relation to the glomerular filtration rate estimated from serum creatinine (eGFR)8, glomerulopathy9, prevalent7 or incident10 CKD, progression to end-stage renal disease10, microalbuminuria11, and the risk of renal cancer12 in relation to air pollutants. These studies enrolled population samples12, stroke patients5, or aged male veterans10. Some studies did not report the level of exposure11, or estimated exposure from the proximity of major roads5, while other findings were representative for exposure levels far above current air quality standards. Other reports focused on coarse particulate7. Of the eight reviewed studies12, none reported on BC exposure. To address this knowledge gap, we analysed data from The Flemish Study on Environment, Genes and Health Outcomes (FLEMENGHO), a family-based population study with catchment area in northern Belgium.Airborne fine particulate (PM2 adrenergic agonists and 80 (37.9%) were on combination therapy with more than one class of blood pressure lowering medication. Intake of statins was reported by 106 participants (12.9%). Serum creatinine and eGFR averaged 86.4 (15.5) µmol/L and 80.9 (16.4) mL/min/1.73m2, respectively and were approximately normally distributed  were women. Age at baseline (2005–2009) averaged (SD) 51.1 (15.6) years . Among 820 participants, 341 (41.6%) had hypertension, of whom 211 (61.9%) were on antihypertensive drug treatment, 75 (5.1%) had a history of cardiovascular disease, and 32 (3.9%) had diabetes. Among 211 patients on antihypertensive drug treatment, 18 (8.5%) took diuretics, 94 (44.6%) inhibitors of the renin system, 17 (8.1%) vasodilators, 2 (0.9%) centrally acting α2.5, respectively. Most clinical and biochemical characteristics were similar across thirds of the BC and PM2.5 distributions, with the exception of plasma glucose which decreased from the lowest to the highest exposure group for both BC (P = 0.027) and PM2.5 (P = 0.033), and the prevalence of smoking (P = 0.027) which was higher in the top PM2.5 exposure group.Tables 3 (0.93–1.55 μg/m3) for BC and 13.1 μg/m3 (12.4–15.3 μg/m3) for PM2.5. While accounting for clustering among study participants sharing a residential address, the levels of the two air pollutants were highly correlated (r = 0.95).The median interval between the assessment of renal function and the midpoint assessment of long-term air pollution (30 June 2012) was 5.6 years  at baseline (2005–2009) and 0.4 years (0.6–2.6 years) at follow-up (2009–2013). Of the 820 participants, 231 lived alone, while 165, 33 and 38 shared a home with two, three or more participants. The median long-term air pollution levels , to which participants were exposed, amounted to 1.10 μg/m2.5, irrespective of whether only the baseline data were used, only the follow-up data, or the baseline and follow-up data (Table P ≥ 0.49). All models accounted for clustering of data among participants living at the same address and were adjusted for sex, age (linear and squared term), mean arterial pressure, heart rate, body mass index, plasma glucose, total-to-HDL cholesterol ratio, γ-glutamyltransferase as biomarker of alcohol consumption, smoking, daily energy expenditure in physical activity, socioeconomic class, and antihypertensive treatment (by drug class). A correlation matrix between continuously distributed covariables measured at baseline was presented in Supplementary Table In multivariable-adjusted cross-sectional analyses of all participants, serum creatinine, eGFR and the prevalence of CKD and microalbuminuria were unrelated to BC and PM-up measurement of renal function. After a median of 4.8 years  serum creatinine increased by 3.7 µmol/L (P < 0.0001) and eGFR decreased by 1.9 mL/min/1.73 m2 (P < 0.0001). From baseline to follow-up, eGFR reverted back to a value > 60 mL/min/1.73 m2 in 7 participants with CKD at baseline and microalbuminuria disappeared in 2. Of the 600 participants with CKD stage ≤ 2 at baseline, 48 progressed to CKD stage ≥ 3 at follow-up (P = 0.0004); among 549 participants without microalbuminuria at baseline, 9 developed microalbuminuria (P = 0.038). In longitudinal analyses (Table 2.5 (Table P ≥ 0.48). The geographical associations of the multivariable-adjusted percent change in the eGFR in relation to BC and PM2.5 appear in Figs. Table P < 0.000 and eGFRP = 0.68) and + 0.20 (P = 0.52) in the individual data and in the data aggregated by municipality  and − 0.022 (P = 0.94), respectively , systolic left ventricular function was slightly worse (P ≤ 0.027) with higher BC and PM2.5 exposure with association sizes per interquartile range increment ranging from − 0.339 to − 0.458% for longitudinal strain and from − 0.033 to − 0.049 s−1 for the longitudinal strain rate13. In the Environmental Influence on Early Ageing study (ENVIRONAGE)14, in 381 mother–offspring pairs, an interquartile range increment in PM2.5 exposure over the entire pregnancy was positively associated with mitochondrial DNA methylation  and inversely associated with mitochondrial DNA content in placental tissue . Taken together, these divergent findings must be interpreted within the context of the circulatory kinetics of the ultrafine particulate. The PM2.5 fraction and the nano-sized BC particles readily penetrate the air-blood barrier in the lung alveoli and are subsequently dispersed body wide, as evidenced by the detection of BC particles in urine4. However, the heart is the first target organ on the circulatory path of these toxic particles. The uterine arteries are tributaries branching off directly from the internal iliac artery. At term, maternal blood flow to the placenta is within the 600–700 mL/min range and the length of the foetal capillaries is close to 320 kilometres15. The renal blood flow represents 20% of cardiac output, but over 90% of the renal blood flow is distributed to the cortex16. These haemodynamic properties, the downstream dilution of the toxic particles from their point of entry into the circulation, and the capillary surface exposed probably explain the divergent vulnerability of organs.In the current population study, we evaluated the associations of eGFR and change in eGFR with BC and PM17. Most previous studies focused on PM1012. In 3901 participants enrolled in the Multi-Ethnic Study of Atherosclerosis, the urinary albumin-to-creatinine ratio was measured at three visits 1.5 to 2 years apart from baseline (2000–2004) and correlated with PM10 as measured by ambient air pollution monitors over 1 month, 2 months and 20 years before the first visit, with adjustments applied for race/ethnicity, sex, age smoking, second-hand smoking, body mass index and dietary protein intake. The study did not demonstrate a significant association between prevalent microalbuminuria or accelerated development of microalbuminuria in relation to PM1011. In contrast, among 1103 consecutive Boston-area patients hospitalised with acute ischaemic stroke between 1999 and 2004, eGFR was correlated with residential distance to major roadways ranging from 50 m or less to over 1 km with adjustments applied for race, sex, age, smoking, comorbid conditions, treatment with angiotensin-converting enzyme inhibitors, and neighbourhood-level socioeconomic status5. Patients living closer to a major roadway had lower eGFR than patients living farther away (P for trend = 0.01). Patients living at a distance of 50 m vs. 1 km had a 3.9 mL/min/1.73 m2 lower eGFR (P = 0.007)5. Among 21,656 residents of Taipei (2007–2009)7, eGFR and the prevalence of CKD (eGFR < 60 mL/min/1.73 m2) were correlated with PMcoarse, PM10 and PM2.5 with adjustments applied for sex, age, body mass index, hypertension , fasting blood glucose, total serum cholesterol, smoking and drinking, educational attainment, and distance to a major roads (continuous). eGFR was 0.69 mL/min/1.73 m2 lower per interquartile range increment in PM10 (5.83 μg/m3), while the odds of having CKD were 1.15 times higher for the same increment in PM107. The corresponding estimates for PMcoarse (6.59 μg/m3) were − 1.07 mL/min/1.73 m2 for eGFR and 1.26 for CKD. In contrast to the associations with PM10 and coarse PM, those with PM2.5 did not reach statistical significance7.Particulate matter is a mixture of solid and liquid particles in the air2.5 reported conflicting results10. In the cross-sectional Taiwanese study reviewed above7, there was no association between eGFR or CKD with PM2.5 . In a longitudinal analysis of 669 men enrolled in the Veterans Administration Normative Aging Study with up to four visits between 2000 and 2011 (n = 1715 visits)6, 1-year exposure to PM2.5 prior to each visit was assessed using a validated spatiotemporal model that utilised satellite remote-sensing aerosol optical depth data. In all person-visits by calendar year, the mean yearly average PM2.5 concentrations increased from 10.5 μg/m3 in 2000 to their highest level of 11.8 μg/m3 in 2002 and later decreased to less than 9.0 μg/m3 in 2010 and 2011. eGFR was analysed by time-varying linear mixed-effects regression models as continuous function of the 1-year PM2.5 levels, while adjusting for a large number of covariables. A 2.1-μg/m3 (interquartile range) higher 1-year PM2.5 was associated with a 1.87 mL/min/1.73 m2 lower eGFR . A 2.1 μg/m3 higher 1-year PM2.5 was also associated with an additional annual decrease in eGFR of 0.60 mL/min/1.73 m2 per year . A recent study linked the Environmental Protection Agency and the Department of Veterans Affairs databases to build an observational cohort of 2,482,737 United States veterans10. It applied survival models to evaluate the association of PM2.5 concentrations and risk of incident CKD and end-stage renal disease10. County-level exposure was defined at baseline as the annual average PM2.5 concentrations in 2004, and as a time-varying variable updated annually and as cohort participants moved. In analyses of baseline exposure , a 10-μg/m3 increase in PM2.5 concentration was associated with a 20% to 30% increase in the risk of CKD and end-stage renal dysfunction. The time-varying analyses produced similar results10. A Chinese study of 71,151 renal biopsies involving 282 cities with standardisation for age and region identified IgA nephropathy (28.1%) and membranous nephropathy (23.1%) as the leading causes of nephropathy9. Three-year average PM2.5 exposure varied among the 282 cities, ranging from 6 to 114 μg/m3 . Each 10-mg/m3 increase in the PM2.5 concentration was associated with 14% higher odds for membranous nephropathy9  in regions with PM2.5 concentration in excess of 70 μg/m3.Human studies relating renal function to exposure to PM18, an approach applied in several previous studies of the renal responses to exposure to PM2.5. Our study is the first to address renal outcomes in relation to BC. Furthermore, our cohort study was population based and therefore representative for exposure, housing conditions and lifestyle in an affluent West-European country. Our study included cross-sectional and longitudinal analyses, both adjusted for a wide range of confounders, in the longitudinal analyses in a time-dependent manner. Sensitivity analyses stratified for various subgroups and minimally adjusted analyses were confirmatory. Notwithstanding these strong points, our study must also be interpreted within the context of its potential limitations. First, the sample size of the study population was relatively small compared with other studies of renal function in relation to PM2.510. Second, due to a change in methodology, serum creatinine was measured by slightly different methods in 536 of 653 participants (82.1%) at baseline and follow-up. However, as reported previously19, the difference in the annual decrease in eGFR between participants who had their serum creatinine measured by different compared with the same method averaged only 0.05 mL/min/1.73 m2 (P = 0.60). Moreover, whatever creatinine assay was used, the annual eGFR decrease was similar as that observed in other European population studies20. Third, as in previous studies of chronic air pollution21, the assessment of renal function and the collection of the air pollution data was not done simultaneously. On the other hand, we used high-resolution spatial data. Several studies in the Netherlands22, Italy (Rome)23, the UK24, and Canada (Vancouver)25 demonstrated that the land use models applied in our current study are representative for the air pollution over periods of 10 years or longer prior to the actual modelling25. Fourth, the incidence of microalbuminuria was low, precluding subgroup analyses for this endpoint. The data given in Table In our current study, we related the renal health outcomes of individual study participants with subject-level estimates of exposure to ultrafine particulate matter. This approach mitigates the so-called ecological fallacy, which arises when aggregate measures of outcome, e.g., cancer incidence by municipality are associated with aggregate estimates of exposure26 and is registered at the Belgian Data Protection Authority . The ethics committee of the University Hospital Leuven, Belgium, approved secondary use of FLEMENGHO data . From August 1985 until November 1990, a random sample of the households, stratified for sex and age  and living in a geographically defined area of Northern Belgium  complies with the Helsinki declaration-up assessment of their renal function on average 4.7 years  later27. Therefore, the number of participants available for the cross-sectional and longitudinal analyses totalled 820 and 653, respectively.Participants were repeatedly followed-up. From 2005 to 2009, we invited 1031 surviving participants, who still resided in the catchment area of the study  and who had not been institutionalised. Of these, 828 renewed informed consent . We excluded 8 participants, because their renal function had not been assessed. Of 820 participants examined at baseline, 653 took part in a followBlood pressure (continuous) was the average of five consecutive auscultatory readings obtained after participants had rested for 5 min in the sitting position. Mean arterial pressure (continuous) was diastolic blood pressure plus one third of pulse pressure (the difference between systolic and diastolic blood pressure). Hypertension  was a blood pressure of ≥ 140 mm Hg systolic, or ≥ 90 mm Hg diastolic, or use of antihypertensive drugs. Antihypertensive drugs were classified  as diuretics , inhibitors of the renin-angiotensin system , and vasodilators . Body mass index (continuous) was weight (kilogram) divided by height (metre) squared.28. Participants with a daily alcohol consumption of 5 g/day or more were categorised as drinkers.The study nurses also administered a standardised questionnaire inquiring into each participant’s medical history, smoking and drinking habits, intake of medications, habitual physical activity during leisure time and at work, and socioeconomic status. Smoking was the current use of any smoking materials on a daily basis . The amount of alcohol consumed expressed in gram per day was computed from the self-reported frequency and type of alcoholic beverages used  and their alcohol content 29, and simplified into a linear scale with scores ranging from 1 to 330. Using validated questionnaires28 and published tables31, we computed the energy spent in physical activity (continuous) from body weight, time devoted to work, walking, sports and leisure time activities, and type of physical activity. This covariable was introduced, because of the known associations of eGFR33 and albuminuria34 with strenuous physical activity.Socioeconomic status was coded according to the complex scales provided by the UK Office of Population Censuses and Surveys-glutamyltransferase . Diabetes mellitus was a fasting glucose exceeding 7.0 mmol/L (126 mg/dL) or use of antidiabetic agents. eGFR was calculated based on the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation35, using age, sex and serum creatinine. The single laboratory involved in measuring the serum creatinine concentration in our study implemented the IDMS-traceable (isotope-dilution mass spectrometry) creatinine assay36, starting from 18 December 2008. Of the baseline creatinine measurements 703 (85.7%) were done before this date. The remaining 117 baseline measurements and all 653 follow-up measurements were done after 18 December 2008. The urine samples were analysed for microalbumin and creatinine. CKD was staged following the National Kidney Foundation (KDOQI) guideline37. Microalbuminuria was defined by an albumin-to-creatinine ratio of at least 3.5 mg/mmol in women or 2.5 mg/mmol in men38. A single blood and urine sample were used to determine the CKD endpoint in categorical analyses, defined as an eGFR of 60 mL/min/1.73 m2, in line with our previous research27 and landmark population studies40.Venous blood samples were obtained after 8 h fasting. Participants collected a 24-h urine sample. After centrifugation and aliquoting, blood-derived specimens were stored at − 80 °C and 10-ml urine samples at − 40 °C. A single certified laboratory did the routine biochemistry, using quality-controlled automated methods. Blood samples were analysed for plasma glucose and total and high-density lipoprotein (HDL) serum cholesterol, serum creatinine and serum γ17. PM2.5 has an aerodynamic diameter of less than 2.5 μm. Upon inhalation, PM2.5 reaches the smallest airways and alveoli and can even cross the blood-air barrier and directly penetrate into the blood stream. BC is a component of PM2.5, consists of pure carbon in several bond forms, and finds its origin in the incomplete combustion of fossil fuels, biofuel or biomass.Particulate matter is the sum of all solid and liquid particles suspended in air2.5 exposure (µg/m3) were calculated for each participant’s residential address at the time of the air quality measurements (2010–2014), using a high resolution spatiotemporal interpolation method42 that takes into account land-cover data obtained from satellite images (CORINE database [http://www.eea.europa.eu/publications/COR0-landcover/at_download/file]) and pollution data of fixed monitoring stations in combination with a dispersion model42. This approach provides daily exposure values in a dense irregular receptor grid, using input from the Belgian telemetric air quality networks and emissions from point and line sources. The air quality network includes 14 monitoring stations for black carbon and 34 for PM2.5. Models covered data averaged over a 5-year period (2010–2014), which reflect the long-term spatial air pollution and are representative for earlier periods25. Model performance was evaluated by leave-one-out cross-validation across monitoring stations. The spatiotemporal explained variance was nearly 80%42 for BC and PM2.5.In the current study, BC and PM2.5 by sorting the measurements from the smallest to the largest and then applying the inverse cumulative normal function. We computed Pearson correlation coefficients between continuously distributed data and Spearman rank correlations to assess the association between geocorrelation between percentage changes in eGFR and the exposure to BC and PM2.5.For database management and statistical analysis, we used the SAS system, version 9.4 . Departure from normality was evaluated by Shapiro–Wilk’s statistic. We normalised the distributions of γ-glutamyltransferase, and the daily energy expenditure in physical activity by a logarithmic transformation. In between-group analyses, means were compared using the large-sample z-test, ANOVA and proportions by Fisher’s exact test. Longitudinal changes were assessed by a t-statistic of the distributions of the follow-up minus baseline values for continuous variables and by the McNemar test for categorical variables. We rank normalised the distribution of BC and PM44. The covariables in the cross-sectional analyses included: sex, age (linear and squared term), mean arterial pressure, heart rate, body mass index, plasma glucose, total-to-HDL cholesterol ratio, γ-glutamyltransferase as biomarker of alcohol consumption, smoking, daily energy expenditure in physical activity, socioeconomic class, and antihypertensive treatment (by drug class). In longitudinal analyses of 653 participants, we accounted for baseline serum creatinine and baseline eGFR by calculating the percent change between the repeat and the first measurement of these biomarkers, while adjusting for sex, baseline age (linear and square term), socioeconomic class, follow-up duration, baseline body mass index, and the baseline value of and change during follow-up in mean arterial pressure, heart rate, plasma glucose, the total-to-HDL serum cholesterol ratio, γ-glutamyltransferase, smoking status, and the intake of antihypertensive drugs . The association of the prevalence  or incidence  of CKD and microalbuminuria with the exposure to particulate were derived by mixed models and expressed as odds ratios, adjusted as outlined above.For the cross-sectional analysis, the association of renal function measures with air pollution was assessed by a generalised linear mixed model adjusting for residential clustering of participants (sharing the same address) as random effect and with adjustments applied for relevant covariablesIn sensitivity analyses, we stratified the participants according to sex, age (< 60 vs. ≥ 60 years), smoking status (non-smokers versus smokers), and the amount of alcohol consumed per day (< 5 vs. ≥ 5 g). In addition, we re-ran the cross-sectional and longitudinal analyses, while adjusting for a reduced set of covariables, thereby removing potential mediators of renal function decline.The Flemish Study on Environment, Genes and Health Outcomes complies with the Helsinki declaration and is registered at the Belgian Data Protection Authority . The ethics committee of the University Hospital Leuven, Belgium, approved the study .Anonymised participants data will be made available upon request directed to the corresponding author. Proposals will be reviewed and approved by the authors with scientific merit and feasibility as sole criteria. After approval of a proposal, data can be shared via a secure online platform after signing a data access and confidentiality agreement. All data will be made available for a minimum of 5 years from the end of the study.Exposure to polluted air, in contrast to smoking, is unintentional and adversely affects health in multiple ways, so that prevention is of paramount importance and global efforts to improve air quality should continue. What our current study adds is that at current exposure levels in a semirural area of Flanders renal function is not associated with ultrafine particulate, including BC, in predominantly healthy people representative of the general population.Supplementary Information.


Obesity, a physiological situation where different proinflammatory cytokines and hormones are secreted, is a major risk factor for breast cancer. Mitochondrial functionality exhibits a relevant role in the tumorigenic potential of a cancer cell. In the present study, it has been examined the influence of an obesity-related inflammation ELIT treatment , which aims to stimulate the hormonal conditions of a postmenopausal obese woman on the mitochondrial functionality and invasiveness of MCF7 and T47D breast cancer cell lines, which display a different ratio of both estrogen receptor isoforms, ERα and ERβ. The results showed a decrease in mitochondrial functionality, with an increase in oxidative stress and invasiveness and motility, in the MCF7 cell line (high ERα/ERβ ratio) compared to a maintained status in the T47D cell line (low ERα/ERβ ratio) after ELIT treatment. In addition, breast cancer biopsies were analyzed, showing that breast tumors of obese patients present a high positive correlation between IL-6 receptor and ERβ and have an increased expression of cytokines, antioxidant enzymes, and mitochondrial biogenesis and dynamics genes. Altogether, giving special importance to ERβ in the pathology of obese patients with breast cancer is necessary, approaching to personalized medicine. Cancer, a pathology characterized by the excessive proliferation of tumor cells, is the second cause of death in the world, responsible for approximately 9.6 million deaths in 2018. In addition, breast cancer is a disease causing more than half a million deaths annually, projecting an increase in its incidence to a total of 3.2 million new cases per year in 2050 [Obesity is a chronic metabolic disease characterized by excess fat accumulation in the body, and its prevalence has increased markedly in the last two decades in the most developed countries . In obesThe cellular effects of estrogens are mediated by their binding to estrogen receptors (ER) alpha (ERα), beta(ERβ), and GPER. Although ERα and ERβ show homology in the DNA and ligand-binding domains, their activity and affinity for 17β-estradiol are different, with ERα having a higher affinity for it. In addition, the expression pattern of the receptors also varies according to the cell type ,9,10. ERAnother adipokine synthesized and secreted by adipocytes is leptin, whose circulating levels are directly correlated with the individual’s BMI . Leptin It is worth noting that obesity is considered an inducing factor of inflammation, where adipose tissue takes on the role of producer of cytokines and inflammatory proteins. However, the biochemical role between inflammation and cancer-inducing cellular modifications has not yet been elucidated . In thisThe expression of inflammatory cytokines, such as interleukin-6 (IL6) and Tumor necrosis factor alpha (TNFα), have been related to an increase in invasiveness and a poor prognosis in breast cancer . IL6 is Mitochondria is a cellular organelle that is highly influenced in response to estrogens, and it is the main source of reactive oxygen species (ROS), playing an important role in tumor processes, as well as in proliferation and apoptosis ,21,22. TMitochondrial function depends on mitochondria morphology, changes in shape, number, and location, which can translate into important functional modifications. Thus, mitochondrial biogenesis is the combination of both proliferation  and differentiation  processes , while mMitochondrial structure and function can be regulated by the activation of ERα and ERβ receptors by binding of 17β-estradiol ,26. RegaDysfunctional mitochondria play a relevant role in cancer and in the epithelial-to-mesenchymal transition (EMT) program in breast cancer . As a coThe aim of the present work was to study mitochondrial functionality and invasiveness, analyzing mitochondrial biogenesis and dynamics processes, as well as oxidative stress and inflammatory status and motility, in cell lines with different estrogen receptors ratio, exposed to a treatment consisting of 17β-estradiol, leptin, IL-6, and TNFα simulating circulating hormonal conditions in a postmenopausal obese woman ,32,33 LiSpecific reagents 17β-estradiol, leptin, interleukin-6, and TNF-α were purchased from Sigma-Aldrich . Dulbecco’s modified Eagle’s medium without phenol red was purchased from GIBCO , Fetal Bovine Serum, and antibiotics solution (penicillin and streptomycin) from Biological Industries . Routine chemicals were supplied by Sigma-Aldrich , Panreac , and Bio-Rad Laboratories .2. To avoid phenol red estrogenic effect, cells were seeded in 6-well or 96-well plates in phenol red-free DMEM containing 10% FBS and 1% antibiotics 24 h prior to treatment. Cells were treated, at 70–80% confluence, with vehicle (0.1% DMSO) or ELIT treatment  for 24 or 48 h.MCF7 and T47D breast cancer cell lines were purchased from American Type Culture Collection ATCC  and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% Foetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) at 37 °C with 5% CO4 cells/well for MCF7 and 3.2 × 104 cells/well for T47D cell line) and treated with ELIT for 48 h. Hydrogen peroxide production was determined by Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit, following the manufacturer’s protocol, as described previously [® Red reagent, following the manufacturer’s protocol, as described previously [Cells were seeded in 96-well plates  and treated with ELIT for 48 h. Cells were stained with 250 nM of Nonyl Acridine Orange (NAO) to measure cardiolipin content as previously described [Cells were seeded in 96-well plates  in 6-well plates and treated with ELIT for 24 h. Total RNA from cultured cells or breast cancer human biopsies (25 mg) was isolated using TRI Reagent , following the manufacturer’s protocol, and then quantified using a BioSpec-nano spectrophotometer  set at 260 nm and 280 nm, getting 260/280 nm ratio. Samples were retrotranscribed to cDNA, and PCR reactions were carried out as previously reported [Cells were seeded . Membranes were blocked in 5% non-fat powdered milk in TBS with 0.05% Tween for 1 h. Antisera against OXPHOS complexes , SOD-1 , SOD-2 , CAT , GRd , 4-HNE , COXIV , PGC1α , and GAPDH  were used as primary antibodies. Protein bands were visualized as described previously by [After 48 h of ELIT treatment, cells were harvested as described previously by Torrens-Mas . Proteinously by .For 4-hydroxy-2-nonenal (4-HNE) adducts analysis, as lipid oxidative damage marker, 20 μg of total protein from cell lysate, processed as previously described by Pons et al. .6 cells/well for T47D cell line and 8.5 × 105 cells/well for MCF7 cell line. Wound healing assay was performed as previously described by Torrens-Mas et al. [Cells were seeded in six-well plates at a density of 1 × 10s et al. . The are5 cells/well for MCF7 and 5 × 105 cells/well for T47D. After 24 h, cells were treated with ELIT treatment for 24 h. Then, cells were incubated with MitoTracker™ Green 0.5 µM  for 1 h and LysoTracker™ Red 0.5 µM  for 20 min, both at 37 °C in the dark. For DNA staining, cells were incubated with 1/200 dilution of 1 μg/mL Hoechst 33342  for 5 min at 37 °C in the dark.Cells were seeded on a glass coverslip inside 6-well plates at a density of 2 × 10The fluorescence was monitored with a Leica TCS-SPE Confocal Microscope, using 63× immersion oil (147 N.A.) objective lens. Fluorescence excitation/emission was 490/516 nm for MitoTracker Green, 577/590 for LysoTracker Red, and 350/455 nm for Hoechst 33342.Mitochondrial Roundness was analyzed in ImageJ software with Mito-Morphology Macro designed by Ruben K. Dagda at the University of Pittsburgh (2010). This macro is currently maintained and supported by grants NIH/NINDS R01NS105783-01 grant and by NIH/NIGMS R25 1R25-OD023795-01.3 cells/well for MCF7 and 9.600 cells/well for T47D into XFe96 well cell culture plates and incubated overnight to allow attachment at 37 °C in 5% CO2. After 24 h, cells were incubated with vehicle or ELIT treatment. After 48 h of incubation, cells were maintained in 200 μL/well of XF assay media at 37 °C, in a non-CO2 incubator for 1 h. During the incubation time, mitochondrial complex inhibitors  were preloaded for OCR measurements, in XF assay media into the injection ports in the XFe96 sensor cartridge.Real-time oxygen consumption rates (OCRs) were determined for MCF7 and T47D cells using the Seahorse Extracellular Flux (XFe96) analyzer . Cells were seeded at a density of 4.8 × 102).Human breast cancer biopsies were obtained from 33 women, ages between 45–90 years. Samples of these patients were obtained from the Biological Specimen Bank of Son Llàtzer Hospital and as specified by and with the necessary permission granted from the Balearic Island Bioethics Committee. Tumor samples were collected immediately after tumor removal and were frozen in isopentane for analysis as described by Sastre-Serra et al. . Writtent-test with statistical significance was set at p < 0.05 (*). The statistical differences in human samples were analyzed using Pearson’s correlation with statistical significance was set at p < 0.01 (**) and p < 0.05 (*).The statistical analyses were performed with the Statistical Programme for the Social Sciences software for Windows . Data are presented as mean ± standard error of the mean (SEM). The statistical differences in cell lines between vehicle- and ELIT-treated cells were analyzed using a Student’s IL6, IL6R, CXCL8, PTGS2, TNF, and STAT3) showed a statistically significant increase in both MCF7 and T47D cell lines after 24 h of ELIT (17β-estradiol (10 nM), leptin (100 ng/mL), interleukin-6 (50 ng/mL), and TNFα (10 ng/mL)) treatment. In contrast, PPARG anti-inflammatory gene had decreased expression in the MCF7 cell line, and TGFB anti-inflammatory gene showed a statistically significant increase in the T47D cell line.To confirm the obesity-related inflammation treatment effectivity, mRNA expression of main inflammatory genes was determined. As shown in 2O2 production increased in both cell lines (+100% in MCF7 and +34% in T47D cells). However, cardiolipin content, as an indicator of mitochondrial inner membrane quantity, decreased in MCF7 treated cells. Moreover, as shown in As shown in SOD2  showed a high increase in both cell lines; in addition, a decrease in SOD1 (copper/zinc superoxide dismutase) was observed. However, ELIT-treated MCF7 cells showed a general decrease in catalase (CAT) and glutathione reductase (GSR). Nevertheless, in the T47D cell line, ELIT treatment increased GSR and nuclear factor erythroid 2-related factor 2 (NFE2L2) expression, as shown in The mRNA expression of antioxidant enzymes was analyzed  in both Main antioxidant protein expression levels were determined  after 48ESRRA) and Twinkle (TWNK) were found in ELIT-treated cells versus non-treated cells. Moreover, uncoupling proteins 2 and 5  mRNA expression was analyzed, and a decrease in UCP2 expression in both cell lines was accompanied by a decrease in uncoupling protein 5 expression in the MCF7 cell line.Mitochondrial biogenesis genes were also checked, as shown in Mitochondrial biogenesis master regulator PPARGC1A protein levels increased after ELIT treatment in the MCF7 cell line. However, to check the effect of ELIT treatment on the mitochondrial respiratory chain (OXPHOS), protein levels of these OXPHOS complexes were analyzed. As seen in To further investigate mitochondrial function, the oxygen consumption rate (OCR) was determined. As shown in MFN1, MFN2, OMA1, OPA1 showed decreased levels in the MCF7 cell line, whereas, in the T47D cell line, ELIT treatment increased expression of MFN1, MFN2, and OMA1. Fission-related mitochondrial genes presented a different pattern expression between cell lines, with a statistically significant increase in FIS1 mRNA expression in the T47D cell line after ELIT treatment.As shown in On the one hand, mitochondrial networking was modified after 24 h-ELIT treatment in both MCF7 and T47D cell lines. However, as shown in CDH1) expression was decreased in MCF7 cells after 24 h ELIT treatment, but Matrix Metalloproteinase 9 (MMP9) expression increased three times with respect to vehicle-treated cells. In contrast, CDH1 expression increased in the T47D cell line after treatment. In addition to these results, a wound-healing assay was performed. As shown in As shown in ESR1) mRNA expression was decreased in both cell lines after 24 h-ELIT treatment. However, estrogen receptor beta (ESR2) and GPER (GPER1) mRNA expression only shown a decrease in the MCF7 cell line, maintaining its expression in the T47D cell line.To observe the effects of obesity-related inflammation treatment over estrogen receptors alpha (ERα), beta (ERβ), and GPER in MCF7 and T47D breast cancer cell lines, mRNA expression was analyzed. As shown in CXCR8, CXCL8, TNF, SLC25A14, NRF1, NFE2L2, PPARGC1A, and FIS1 were significantly and positively correlated with IL6R expression in the nw group. CXCR8, TNF, SLC25A14, NRF1, NFE2L2, and PPARGC1A were significantly and positively correlated with IL6R expression in the ow group. CXCR8, CXCL8, TNF, PTGS2, ESR2, GPX1, SOD1, SLC25A14, NRF1, NFE2L2, PPARGC1A, SIRT1, FIS1, and OMA1 were significantly and positively correlated with IL6R expression in the ow group.ESR1 and ESR2 gene expression correlations are shown in IL6, CXCL8, SOD1, and SSBP1 were significantly and negatively correlated with ESR1 expression; instead, SIRT3 was significantly and positively correlated. On the other hand, IL6R, CXCR8, GPX1, SLC25A14, NRF1, PPARGC1A, SIRT1, and OMA1 were significantly and positively correlated with ESR2 expression.Moreover, In this study, the effects of obesity related-inflammation on mitochondrial functionality in breast cancer cell lines and breast tumors, focusing on estrogen receptors ratio, were analyzed. Moreover, invasiveness in this situation was also analyzed. We demonstrated that ERβ, in an inflammatory and obesity condition, maintains mitochondrial functionality and avoids invasiveness in breast cancer cell lines. Moreover, we found a strong correlation between interleukin-6 receptor gene expression and inflammation, mitochondrial functionality, and oxidative stress markers, as well as with estrogen receptor beta, in breast cancer human samples in different BMI situations. Obesity stimulates the adipose tissues to release inflammatory mediators such as tumor necrosis factor α and interleukin 6, predisposing them to a proinflammatory state and oxidative stress ,39. ThesPrevious studies in our group and other research groups have shown the independent effects of 17β-estradiol or leptin on oxidative stress and mitochondrial biogenesis and dynamics ,42,43,442O2 and superoxide anion, as similarly described by some authors [In this study, MCF7 and T47D cell lines have been treated with an ELIT inflammatory cocktail with the aim of generates an inflammation situation (IL6 and TNFα) in the presence of Leptin and 17β-estradiol levels, simulating the physiological condition of postmenopausal obese women. In this way, an increase in reactive oxygen species has been observed in both cell lines; however, in the T47D cell line, a lower increase in the production of hydrogen peroxide and levels of superoxide anion was observed. These data make more sense when studying the oxidative damage present in both cell lines, where a significant increase was only observed in the MCF7 cell line. On the other hand, it seems that the T47D treated cell line does not present differences compared to the control group, despite presenting higher levels of H authors ,45,46.NFE2L2, a transcription factor that controls mainly antioxidant enzymes expression, and glutathione reductase, which recycles glutathione, was also increased, avoiding a high increase in free radical levels in low ERα/ERβ ratio T47D cell line. In addition, it should be noted that those tumors with a high correlation between IL6R and ESR2 gene expression presented an increase in antioxidant enzymes, thus could palliate levels of free radicals, which can ultimately diminish oxidative damage.Oxidative stress is generated in breast cancer, and in many other pathologies, in two main ways. The first one, as observed in the high ERα/ERβ ratio MCF7 cell line after ELIt treatment, is a decrease in the expression of the antioxidant enzymes. It was observed that all antioxidant enzymes had decreased their gene or protein expression in the MCF7 cell line, except the SOD2 enzyme that had increased expression in both MCF7 and T47D cell lines. It has been described that SOD2 plays a role as a free radical detector, increasing gene and/or protein expression in order to alleviate oxidative damage [The other pathway that increases oxidative stress is the poor maintenance of a functional mitochondrial pool . To achiESRRA) and Twinkle (TWNK). ESRRA belongs to a superfamily of nuclear receptors independent of estrogen activation. Its expression is induced and activated by PGC1α, and the two factors together are capable of binding to response elements and promoting the initiation and elongation of the transcription of metabolic and mitochondrial target genes [However, the T47D cell line, with high levels of ERβ, had mitochondrial dynamics elevated, maintaining a more functional pool of mitochondria. In the case of mitochondrial biogenesis, it should be noted that there are two genes that presented a very significant elevation, estrogen-related receptor alpha (et genes . Twinkleet genes . In factet genes . Taking into account that patients with tumors with low ERα/ERβ ratios have a worse response to chemical agents oxidative damage inductors , we obseIn addition to mitochondrial biogenesis and dynamics, our study reveals an oxygen consumption rate decreased in the MCF7 cell line that supports all the results commented above. It is worth noting that ELIT treatment decreases basal respiration, maximal respiratory capacity, ATP-linked respiration as much as proton leak in the MCF7 cell line. Proton leak decrease is in concordance with low uncoupling proteins mRNA expression levels found in the MCF7 cell line after ELIT treatment. UCPs, which promote proton leak across the inner mitochondrial membrane, have emerged as essential regulators of mitochondrial membrane potential, respiratory activity, and ROS generation . As seenOxidative stress, inflammation, and poor mitochondrial network observed in MCF7 cell lines after ELIT treatment leads to a worse situation, and some authors have described a relationship between this situation and metastasis . Our resAs mentioned above, the MCF7 cell line has a higher ERα/ERβ ratio, and the T47D cell line has a lower ratio. ELIT-treatment decreases estrogen receptor alpha mRNA expression in both MCF7 and T47D cell lines, as shown in results. However, estrogen receptor beta mRNA expression only decreases in the MCF7 cell line but not in the T47D cell line. This fact supports that all the results showed in our study could be through estrogen receptor beta T47D cell lines maintenance, giving to this receptor subtype the protective role that previously some studies have been described ,39. The presence of estrogen receptor beta allows maintaining a more functional mitochondrial pool, with active mitochondrial biogenesis and dynamics, which means less production of reactive oxygen species and better mitochondrial metabolism in an obesity-related inflammation condition. In addition, antioxidant enzymes are active, preventing oxidative damage and, at least in part, invasiveness.This study could be important to remark the importance of estrogen receptor beta and mitochondria as an important organelle in the development and prognosis of breast cancer in obese patients. Likewise, more studies are necessary in order to clarify the estrogen receptor beta mechanism in breast cancer and establish it as a clinical biomarker, as is the estrogen receptor alpha.


We also found that p32/C1qbp-deficient DCs exhibited impaired production of IL-1β, IL-23, and mitochondrial reactive oxygen species (mtROS) after IMQ stimulation. Because the inhibition of mtROS suppressed IMQ-induced DC activation and psoriatic inflammation, we presume that p32/C1qbp and mtROS can serve as therapeutic targets in psoriasis.Psoriasis is a common immune-mediated, chronic, inflammatory skin disease that affects approximately 2–3% of the population worldwide. Although there is increasing evidence regarding the essential roles of the interleukin (IL)-23/IL-17 axis and dendritic cell (DC)-T cell crosstalk in the development of skin inflammation, the contributions of mitochondrial function to psoriasis are unclear. In a mouse model of imiquimod (IMQ)-induced psoriasiform skin inflammation, we found that hematopoietic cell-specific genetic deletion of p32/C1qbp, a regulator of mitochondrial protein synthesis and metabolism, protects mice from IMQ-induced psoriatic inflammation. Additionally, we demonstrate that p32/C1qbp is an important regulator of IMQ-induced DC activation, both  Psoriasis is a skin disease that affects approximately 2–3% of the world population, including 10% of the population in some Northern European countries . PsoriasPsoriasis is a disorder of both innate and adaptive immune systems, in which dendritic cells (DCs), keratinocytes, and T cells have central roles. DCs are key sentinels of the immune system, which bridge the gap between innate and adaptive immunity. Thus far, several studies have indicated that DCs contribute to the initiation of psoriasis through antigen-presenting molecules , cytokines , and chemokine receptors , 14. Thede novo psoriasis lesions and exacerbate preexisting psoriasis lesions (in vitro (Imiquimod (IMQ) is a TLR 7/8 agonist that has been widely used for the topical treatment of human papilloma virus infection , 24, 25. lesions , 26. In  lesions , 28. Bec lesions , 28, thein vitro , 30.The complement component 1q subcomponent binding protein C1QBP/p32/HABP1) was originally identified as a protein associated with the essential splicing factor ASF/SF2 . Our gro/HABP1 wavia DCs and T cells). Therefore, we developed hematopoietic cell-specific p32/C1qbp-deficient mice. In the present study, we show that p32/C1qbp is involved in psoriasis pathogenesis through DC activation.Various genetic, environmental, and immunological factors reportedly contribute to psoriasis development , 38, 39.flox/flox mice have been described previously (flox/flox Vav1–iCre+ (p32 cKO) and control littermate p32flox/flox Vav1–Cre– (WT) mice were used in this study (C57BL/6 mice were purchased from Japan Clea. Vav1–iCre mice (cat. no. 008610) were obtained from Jackson Laboratory. p32eviously . Age- anis study . All micBone marrow DCs (BMDCs) were prepared as described elsewhere . To geneIMQ-induced psoriasis-like inflammation was elicited and skin severity was assessed as previously described . For theSkin sections were stained with H&E. Epidermal thickness was determined by measuring the mean interfollicular distance under the microscope. For IHC staining, skin cryosections were fixed, blocked, and stained with purified rabbit-anti-mouse Ki-67 Ab (1:200 dilution), followed by anti-rabbit IgG secondary antibody (1:200 dilution). Slides were developed with AEC substrate solution (Vector Laboratories) and then counterstained with hematoxylin. Images were acquired at 200× magnification using a Keyence BZ-X800 microscope.After stimulation or treatment with IMQ (5 μg/mL), BMDCs and mouse ears were collected and washed in PBS, then resuspended in RLT buffer. Total RNA was extracted from BMDCs and mouse ears using an RNeasy Mini Kit (Qiagen), in accordance with the manufacturer’s instructions. RNA concentration (ng/μL) and sample purity (260/280 ratio) were measured using the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific). Reverse transcription of approximately 500 ng of total RNA was performed with PrimeScript RT reagent Kit , in accordance with the manufacturer’s protocol. Gene expression was measured by SYBR Green-based qPCR. Relative quantification was performed using the comparative cycle threshold (Ct) method, relative to 18S ribosomal RNA. PCR primers are listed in Mouse ears were collected and split into dorsal and ventral halves. They were then floated for 40 min at 37°C on the surface of 0.5% trypsin (w/v), which facilitated separation of the dermis from the epidermis. The dermis was cut into small pieces and placed into digestion solution containing collagenase type IV (1.5 mg/mL). Digestion was performed for 90 min at 37°C with mixing. After the completion of digestion, the solution was mixed thoroughly and filtered through a 70-μm nylon filter to obtain a single-cell suspension. For analysis of surface markers, cells were stained in PBS containing 2% (v/v) fetal bovine serum with cell surface antibodies. For intracellular staining with antibodies, cells were fixed with 4% (wt/vol) paraformaldehyde  and permeabilized with saponin (0.5% saponin and 0.5% BSA in PBS) for 15 min at room temperature.BMDCs were stimulated with IMQ (5 and 25 µg/mL) with or without MitoQ (5 and 10 µM). Reagents were added to cells, simultaneously with imiquimod. Antibodies are listed in Whole skin cell suspensions from the ears of WT or p32cKO mice were cultured in cRPMI in the presence of mouse IL-23 , IL-1β , or IL-23 and IL-1β for 72 h. Brefeldin A  was added 2–3 h before intracellular staining and flow cytometry.Whole skin cell suspensions from the ears of WT or p32cKO mice treated with IMQ for 4 consecutive days were re-stimulated with phorbol myristate acetate  and ionomycin  in the presence of brefeldin A  for 2–3 h. They were then stained by the intracellular staining procedure as described to determine IL-17A expression .BMDCs were incubated with 1 µM MitoSOX Red mitochondrial superoxide indicator (Thermo Fisher Scientific) at 37°C for 15 min in HBSS, washed two times with HBSS, and then analyzed with flow cytometry.BMDCs were incubated in HBSS with 5 or 25 µM IMQ for 10–15 min at 37°C. MitoSOX Red , MitoTracker Green FM , and TMRM  were respectively added at the beginning of incubation. BMDCs were washed two times with HBSS, then analyzed with flow cytometry.BMDCs were analyzed using an XF-24 Extracellular Flux Analyzer (Seahorse Bioscience) . BrieflyFor direct immunoblotting, BMDCs were washed in PBS, then lysed with cell lysis buffer . The lysate was eluted with sample buffer and boiled for 5 min at 97°C. SDS-PAGE was performed with 8%–15% polyacrylamide gels using a constant current of 20 mA. The gels were transferred to Immobilon-P membranes (Merck Millipore) using a semidry blotting device for 15 min, with a constant current of 2.5 A. The membranes were blocked with Blocking One  and incubated with the appropriate primary antibody overnight at 4°C in Can Get Signal Solution 1   induced psoriatic inflammation with marked erythema, scaling, and stiffness  in ears from p32cKO mice  in an NF-κB or interferon regulatory factor (IRF)-dependent manner, following IMQ stimulation. Consistent with our in vivo data (–/– DCs)  and tricarboxylic acid (TCA) cycle (citric acid and isocitric acid) were significantly reduced in p32–/– DCs before IMQ stimulation  , 45–47. Previous studies showed that IL-1β is involved in the pathogenesis of psoriasis and psoriatic arthritis , 51, 52.in vitro  can be found in the article/The animal research protocols were approved by the Committee of Ethics on Animal Experiments, Faculty of Medical Sciences, Kyushu University.KG and DK designed the study. SM, KG, YN, DS, and YT carried out the experiments. SM and KG prepared the figures and wrote the manuscript. SO and DK supervised the experiments. KG, SO, and DK reviewed the experiments. All authors contributed to the article and approved the submitted version.This work was supported by JSPS KAKENHI Grant Numbers JP18K11077 and JP16K19196 (both to KG) and JP20H00530 and JP17H01550 (both to DK). This work was also supported by grants from the Takeda Science Foundation, The Shin-Nihon Foundation of Advanced Medical Research, and Charitable Trust Laboratory Medicine Research Foundation of Japan (to KG).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In summary, our study uncovers a critical role of SIRT1-dependent autophagy signaling pathway in ECM protection and thus in the establishment of therapeutic effect of PEMF on IVD degeneration.Intervertebral disc (IVD) degeneration is regarded as a major contributor to low back pain (LBP), causing serious economic burden on individuals and society. Unfortunately, there are limited effective treatment for IVD degeneration. Pulsed electromagnetic field (PEMF) is an economical and effective physical therapy method, with reduced side-effects. It offers certain protection to a number of degenerative diseases. Therefore, understanding the underlying mechanism of PEMF on IVD is important for improving the PEMF therapeutic efficiency. In this study, PEMF up-regulated extracellular matrix (ECM) related genes in degenerated nucleus pulposus (NP) cells. It also increased SIRT1 expression and promoted autophagy in degenerated NP cells. In contrast, the autophagy suppressor 3-methyladenine (3-MA) reversed the beneficial effect of PEMF on ECM production. Similarly, the SIRT1 enzyme activity suppressor EX 527 also inhibited the effect of PEMF on autophagy and ECM production in NP cells, thereby suggesting that PEMF regulated ECM related genes expression through SIRT1-autophagy signaling pathway. Lastly, PEMF significantly reduced IVD degeneration in a rat model of IVD degeneration  Low back pain (LBP) is an age-related condition, the prevalence and impact of which are increasing year by year . In a rein vitro and in vivo (in vitro (Pulsed electromagnetic field (PEMF), as an economical and non-invasive form of physical therapy, with reduced side-effects. It has been used in clinical practice since the 1970s, mainly for the treatment of delayed fracture healing, nonunion, and osteonecrosis . Numerou in vivo . Recent in vitro . In addiin vitro . Althougvia SIRT1-mediated autophagy in cellular and animal models.Interestingly, recent studies have uncovered that PEMF show a potential role in regulating silent information regulator 1 (SIRT1), a NAD + -dependent deacetylase, which might activate autophagy in several tissues . Autophain vitro and in vivo studies, which was confirmed by the oscilloscope and Gaussmeter test.The PEMF stimulation system , Xi’an, China; China Patent no. ZL02224739.4) consisted of a PEMF generator and Helmholtz coils . In cell3, before digestion for 30 min in 0.2% pancreatin. They were then rinsed thrice in PBS and incubated in 0.25% type II collagenase for 4 h, with shaking every half an hour, before terminating digestion. Any undigested tissue was then filtered with a 70-micron filter, before resuspending the digested tissue in DMEM/F12 medium with 10% fetal bovine serum and antibiotics, prior to culture in a 5% CO2 humidified incubator at 37°C. Cells received PEMF treatment 4 h per day, and samples were collected at 0, 24, 48, and 96 h to test apoptosis rate. In order to examine PEMF-mediated autophagy via SIRT1, cells were pretreated with either 10 mM 3-MA (an autophagy suppressor) or EX-527 (a SIRT1 enzymatic activity suppressor) for 2 h before the first PEMF stimulation and then maintained in the same media for the entire experiment. All experiments were repeated thrice for good measure.Our work received ethical approval from the Xijing Hospital, and we received informed consent from all eligible participants. Human intervertebral disc tissue was obtained from patients with lumbar disc herniation  and fresh NP tissue samples were obtained during the surgery. Samples were washed thrice with normal saline, and cut into 2 mm4/ml). In 24, 48, and 96 h timepoint, each well was washed with PBS and then treated using 10-µl of Cell Counting Kit 8  for1 h at 37°C. The optical density was then evaluated via a microplate reader  at 450 nm absorbance.NP cells were seeded to the 96-well plate  and a BioRad CFX96 PCR System . GAPDH served as an endogenous control. The primers used for qRT-PCR reactions were as follows: collagen II (F)5′-CGAGGCAGACAGTACCTTGA-3′, (R) 5′-TGCTCTCGATCTGGTTGTTC-3’; MMP3 (F) 5′-GCTCATCCTACCCATTGCAT-3′, (R) 5′-GCTTCCCTGTCATCTTCAGC-3’; GAPDH (F) 5′-GGCACAGTCAAGGCTFAGAATG-3′, (R) 5′-GGTGGTGAAGACGCCAGTA-3’.Total RNA was extracted from NP cells using the Total RNA Mini Kit , following kit directions, then converted to cDNA, prior to qPCR analysis via SDS-PAGE, before transfer to PVDF membranes (Thermo Fisher Scientific). Next, they were blocked using 5% non-fat milk in TBS-Tween (TBST) at room temperature (RT) for 1 h, before exposure to primary antibodies ) at 4°C overnight (O/N). They were next exposed to secondary HRP-linked antibodies, either goat anti-rabbit or goat anti-mouse secondary antibodies  at a 1:2,000 dilution for 1 h, before protein band detection using chemiluminescence (Bio-Rad).NP cells were thrice rinsed in PBS, before harvest using RIPA buffer and centrifugation at 12,000 g for 15 min. Protein quantification was done with a BCA analysis kit. Proteins were electrophoresized NP cells underwent fixation in 4% paraformaldehyde for 10–15 min, and permeabilization in 0.2% Triton X-100 for 20 min, before blocking in 10% normal goat serum at RT for 1h, and O/N incubation in antibodies against collagen II (1:200) and MMP-3 (1:150) at 4°C. Then, they were thrice washed in PBS before exposure to secondary antibodies  for 1 h at RT, and to DAPI for 10 min. Cells underwent three PBS washes for 15 min after every step. Lastly, they were visualized under a fluorescence microscope and analyzed using the ImageJ software.4, before receiving gradient dehydration in acetone, followed by embedding, and slicing into 70 nm ultrathin sections using LKB-V ultrathin microtome. Subsequently, they were post-stained with uranyl acetate and lead citrate, and visualized with TEM .A TEM  was employed for calculating autophagic vesicles within NP cells. In short, NP cells underwent O/N fixation in 2.5% glutaraldehyde, followed by a 1 h post-fixation in 2% OsOAll animal experiments abided by the Institutional Ethical Committee of Fourth Military Medical University guidelines. Fifteen rats were arbitrarily separated into three groups: control (n = 5); needle puncture (n = 5); and needle puncture and PEMF treatment (n = 5). All rats were intraperitoneally anesthetized with pentobarbital solution . As described in previous studies, following tail skin disinfection with iodophor and alcohol, a 20-gauge needle was used to fully puncture the tail disc from Co6-Co8 levels, including two IVDs (Co6-Co7 and Co7-Co8) . The neeMRI images were captured 8 weeks after treatment, using an MRI system . The IVD degeneration score was computed by two independent spinal surgeons, based on the Pfirrmann grading system, ranging from grade I to grade V.Upon fixation in 4% paraformaldehyde, the IVD tissue was decalcified, paraffin-embedded, and sliced into 7 μm thick sagittal sections, then stained with Hematoxylin and Eosin (H&E), Safranin O-Fast Green (SOFG), and Sirius Red. The histological scores, including NP morphology and cellularity, AF organization, matrix structure and CEP integrity, were determined by independent researchers who were blinded to the study, as reported previously .p < 0.05 was deemed significant.All data were analyzed using one-way ANOVA from the SPSS 13.0 software  and expressed as means ± standard deviations (SDs). Tukey’s post hoc and Dunnett’s tests were employed for significance comparisons . To examine the PEMF-mediated effect on NP cell apoptosis, viability and cell morphology, we stimulated NP cells with PEMF 4 h/day for four consecutive days, and monitored cell apoptosis and viability using flow cytometry and CCK-8 assay at 0, 24, 48, and 96 h. Based on our results, PEMF did not exert any obvious cytotoxicity on NP cells . Moreovein vitro.We also assessed the PEMF-mediated regulation of NP cell ECM related genes expression. Following 4 days of treatment, RT-qPCR data revealed that PEMF markedly enhanced collagen II expression, while decreasing Mmp3 levels in NP cells . ResultsTo assess whether PEMF regulates NP cell autophagy, we examined LC3B and p62/SQSTM1 levels in NP cells, using western blot. As depicted in Next, we examined whether autophagy regulates the PEMF-mediated regulation of ECM related makers expression in NP cells. As illustrated in It is well known that Sirt1 is an upstream regulator of autophagy. Interestingly, we found that PEMF treatment markedly increased Sirt1 expression . We, nexin vivo, we established an IVD degeneration model by puncturing the IVD of rat tail. Firstly, we measured the changes in the weight of the rats to monitor whether PEMF would affect other tissues in rats. No significance change was observed in the weight of rats in each group at 2w, 4w and 8w timepoint with or without PEMF stimulation, which indicates that PEMF is safe and has no side effects in rats , while reducing MMP3 (related to ECM catabolism) levels. However, no significance change was observed in cell characterizations, including cell apoptosis, viability and cell morphology. Even though the positive effect of PEMF in degenerative IVD cells was demonstrated, the role of PEMF in non-degenerated cells remains to be studied. In in vivo studies, we successfully constructed a rat tail IVD degeneration model. Then, we applied PEMF systematically to the rats. To better understand the systemic effects of PEMF on rats, we measured body weight of rats at 2w, 4w and 8w timepoint, and no significance change was observed in each group, which confirmed that PEMF might be a safe noninvasive therapy. It has been shown that PEMF modulates biological functions of many tissues under pathological conditions, including injuries in bones, tendons, muscles and nervous system . Moreover, these effects were reversed by 3-MA administration. Recently, studies have shown that various matrix molecules, including glycosaminoglycans, collagen, constituting the intervertebral disc and cartilage ECM, regulate autophagy through mTOR signaling (Autophagy is an evolutionarily conserved modulatory behavior that sustains cell homeostasis proteins . Previouproteins . Therefoproteins . Anotherproteins . In ordeignaling . Moreoveignaling . Given tin vitro studies. Based on results, EX-527 has no effect on SIRT1 expression, but can severely reduce its enzymatic activity. We demonstrated that EX-527 effectively inhibits PEMF-induced autophagic activation, along with collagen II synthesis and MMP3 decomposition. Using an IVD rat model, we also validated that PEMF successfully improves puncture-stimulated IVD degeneration by elevating SIRT1 levels and activating autophagy. Although previous reports hold varying opinions on PEMF and SIRT1 regulation (In our earlier work, we demonstrated a negative correlation between SIRT1 expression and IVD degeneration . In ordegulation , our resIn conclusion, our research provides evidence that PEMF promotes the SIRT1-autophagic network and reduces IVD degeneration in cells and animal models. These results provide new insights of PEMF in treating IVD degeneration and highlight their therapeutic potential in IVD degeneration. Hence, it is our hope that the present study will promote future application of PEMF as a potential therapeutic treatment for preventing IVD degeneration in clinical use.


The phagocytosis checkpoints of CD47/SIRPα, PD1/PDL1, CD24/SIGLEC10, and MHC/LILRB1 have shown inhibited phagocytosis of macrophages in distinct tumors. However, phagocytosis checkpoints and their therapeutic significance remain largely unknown in intrahepatic cholangiocarcinoma (ICC) patients.We analyzed sequencing data from the Cancer Genome Atlas (TCGA) and identified differently expressed genes between tumors and para‐tumors. Then, we investigated the expression of CD68, SIRPα, PD1, and SIGLEC10 by IHC in 81 ICC patients, and the clinical significance of these markers with different risk factors was also measured.+ or PD1+ TAMs were observed in the tumor tissues of ICC patients with HBV infections compared to non‐HBV‐infected patients. Multivariate analysis indicated that SIRPα and PD1 expression are independent indicators of ICC patient prognosis.Tumor infiltration immune cells analysis from the TCGA data revealed that macrophages significantly increased. Further analysis showed that M0 macrophages were significantly higher and M2 macrophages were significantly lower in ICC compared with paracancerous tissues, while there was no significant difference in M1 macrophages. We then examined some of M1 and M2 markers, and we found that M1 markers  increased, while M2 markers (ARG1 and CD206) decreased in ICCs compared with paracancerous tissues. Furthermore, the expression of CD68, SIRPα, PD1, and SIGLEC10 increased significantly, but LILRB1 expression did not. We also examined the expression of CD68, SIRPα, PD1, and SIGLEC10 in 81 ICC patients by IHC, which revealed a similar expression pattern to that which emerged from the TCGA data. Upon analyzing the correlation between these markers and the progression of ICC patients, we found that the high expression of CD68, SIRPα, and PD1 are correlated with poor progression among ICC patients, while SIGLEC10 shows no correlation. More SIRPα+ TAMs in tumor tissues are negative prognostic markers for ICCs after resection. Furthermore, anti-CD47 in combination with anti-PD1 or CD47/PD1 bispecific antibody (BsAb) may represent promising treatments for ICC. Further studies are also required in the future to confirmed our findings.Hyperactivated CD47/SIRPα and PD1/PD‐L1 signals in CD68 Intrahepatic cholangiocarcinoma (ICC) is an aggressive and invasive malignant tumor occurring within the liver, and it represents the second most common primary malignancy of the liver . In the + T cells and its therapeutic and prognostic significance [The tumor microenvironment is complex and is regulated by multiple immune cells . It playificance , 19. Addificance , 20, 21.+TAMs in patients diagnosed with ICC. Our study highlights that the phagocytosis checkpoints of CD47/SIRPα and PD-1/PD-L1 axes are highly correlated with poor progression among ICC patients, while those of the CD24/SIGLEC10 and MHC-I/LILRB1 axes are not. Therefore, anti-CD47 in combination with anti-PD1 or CD47/PD1 bispecific antibodies (BsAbs) may present a novel and promising treatment option for ICC. Further studies are needed to confirm these findings.In the present study, we first analyzed the various transcriptional expressions of ICC and paracancerous tissue published by previous studies available on the Cancer Genome Atlas (TCGA) databases. The cell type proportion were also examined. And then, we investigated the clinical relevance and prognostic significance of phagocytosis checkpoints expressed by CD68P-value was analyzed to correct for false positive results in TCGA or GTEx. “Adjusted P < 0.05 and Log2 (Fold Change) > 1 or Log2 (Fold Change) <  − 1” were defined as the thresholds for the screening of differential mRNA expression. To further confirm the underlying function of potential targets, the data was analyzed via functional enrichment. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis is a practical resource for analytical study of gene functions and associated high-level genome functional information. To better understand the carcinogenesis of mRNA, the ClusterProfiler package (version: 3.18.0) in R was employed to analyze to enrich the KEGG pathway.RNA-seq was analyzed as our previously described –24. BrieThirty-six ICCs from TCGA datasets were included in this study. Overall survival time (OS) was compared between the high and low TAM-related markers by Kaplan–Meier analysis as previously described , 27. TheCIBERSORT, QUANTISEQ, and MCPCOUTER, which was used to analyze the immune cells infiltration in ICCs as previously described –31. The The sample consisted of 81 consecutive ICC patients who underwent curative resection from June 2, 2016 and December 30, 2019 at the affiliated cancer hospital of Zhengzhou University. This study was approved by the Ethics Committee at the affiliated cancer hospital of Zhengzhou University. All methods and procedures associated with this study were conducted in accordance with the Good Clinical Practice guidelines and accorded with the ethical principles of the Declaration of Helsinki as well as local laws. All enrolled patients were pathologically diagnosed with ICC and were not administered any anti-cancer treatments before surgery. After surgery, follow-ups were made on the patients to examine tumor status every 3 months for the first 2 years and every 6 months from year 3 to year 5. After 5 years, follow-ups were performed every year. The last follow-up took place on October 1, 2021.2O2 for 5 min. After washing with Tris-buffered saline (TBS) containing Tween, endogenous biotin was inhibited by sequential incubation with 0.1% anti-biotin protein and 0.01% biotin , respectively, for 10 min at room temperature. Other non-specific binding sites were blocked with 3% skimmed milk powder for 30 min at room temperature. The tissue section of the intrahepatic cholangiocarcinoma tissue and paracancerous tissue were incubated with the monoclonal mouse antibody anti-human CD68 , SIRPα , PD1, and SIGLEC10  for one night at 4 ℃. Subsequently, the sections were serially rinsed and incubated with second antibodies. Immunohistochemical staining was evaluated independently by two experienced pathologists blinded to the patients’ clinical characteristics and outcomes. A histochemistry score (H-score) based on a combination of the percentage of positive stained cells and their staining intensity was calculated for the semiquantitative analysis as previously described [Formalin-fixed and paraffin-embedded sections of intrahepatic cholangiocarcinoma tissue and paracancerous tissue (5 μm thick) were dewaxed and rehydrated. Antigen retrieval was performed by heating the slides in 10 mM Tris buffer with 1 mM EDTA (pH 9) in a streamer for 20 min. Inhibition of endogenous peroxidase activity was achieved by immersion in 3% Hescribed –34. H-ScFollow-up and survival analysis of ICC Patients were consistent with previous studies , 35. Aftt-test. DFS and OS were calculated using the Kaplan–Meier estimator. Univariable and multivariable Cox proportional hazards regression models were used to estimate hazard ratios along with associated confidence intervals and p-values. Student’s t-test and chi-square (χ2) test were employed for inferential statistical analysis. For all data, P < 0.05 was used to indicate a statistically significant difference.GraphPad Prism 9.0 software  and SPSS® 24.0 software were used to perform the statistical analysis. Quantification of CD68, SIRPα, PD1, and SIGLEC10 density were analyzed via To begin this study, we first retrieved the transcriptome profiling data of ICC patients from the Cancer Genome Atlas (TCGA) database. Nine normal and 36 tumor samples were included. Next, the data was analyzed using R software to study the differential expression of mRNAs. A total of 16,897 genes were identified, and 10,196 were distinguished as differentially expressed mRNAs (DEmRNAs)  signature in ICCs using three methods, CIBERSORT, QUANTISEQ, and MCPCOUTER as previously described –31. MCPCWe examined the prognostic impact of differentially expressed M1, M2 markers and phagocytosis checkpoints in cancer tissues in ICC patients from the Cancer Genome Atlas (TCGA) database. The Kaplan–Meier survival analysis and log-rank test were used to compare the survival rates between high expression (N = 18) and low expression (N = 18) groups of ICC patients. Interestingly, the Kaplan–Meier analysis indicated that patients exhibited a similar survival time based on the expression levels of iNOS, TNF, IL12A, IL12B, ARG1, CD206, CD68, SIRPα, PDCD1, and SIGLEC10 Fig. A–C.Fig. Given that the TCGA database had limited data on ICC patients, we sought to assess the role of “don’t eat me” molecules by using data from additional patients in China. A total of 81 patients between June 2, 2016 and December 30, 2019 were used in this study, including 47 (58.02%) males and 34 (41.98%) females. The median age was 61 years. All the patients received surgery. Prior to surgery, 20 (24.69%) patients had HBV infections, 10 (12.35%) patients had cirrhosis, and two patients had a history of hepatitis C infections. A total of 62 (78.48%) patients had elevated serum CA19-9, seven patients had elevated serum CEA (8.64%), and 55 (67.9%) patients had elevated serum CA724. Additionally, 69 patients had elevated serum total bilirubin, 72 patients had elevated serum direct bilirubin, and 49 patients had elevated serum indirect bilirubin. Moreover, 56 patients had elevated serum alanine transaminase (ALT), and 50 patients had elevated serum aspartate transaminase (AST). Finally, 14 patients were classified as Child–Pugh grade A, and 67 patients were Child–Pugh grade B.Regarding tumors, all patients had a solitary tumor, and seven patients had larger tumors (> 5 cm). A total of 54 patients were TNM stage I or II, while 27 patients were TNM stage III. In addition, 54.32% of tumors had high to intermediate histopathological grading, and 45.68% of tumors had intermediate to low histopathological grading. In addition, 19.75% of patients had lymph node invasion, and 20.99% of patients had microvascular invasion. Patients’ detailed clinicopathologic characteristics are showed in Table To further confirm the expression pattern of SIRPα, PD1, and SIGLEC10 in TAMs in ICCs, 81 ICC tissues and 81 paracancerous tissues were obtained from our hospital. First, H&E staining was performed to prove that the tissues taken were indeed paracancerous and cancerous. Then, immunohistochemistry (IHC) was performed with anti-CD68, anti-SIRPα, anti-SIGLEC10, and anti-PD1 antibodies to detect the phagocytosis checkpoints expression pattern in ICCs. IHC images of representative CD68, SIRPα, SIGLEC10, and PD1 from cancerous and paraneoplastic tissues are shown in Fig. high, SIRPαhigh, and PD1high) and low expression subgroups . It is worth noting that high CD68 expression as well as high SIRPα were positively correlated with high TNM stage, lymph node invasion, high Child–Pugh stage, microvascular invasion, and HBV infection . The significance of PD1 expression in T cells among ICC patients has been reported in previous studies [The final follow-up occurred on October 1, 2021. Up to October 1, 2021, all the patients died. The 1-year and 5-year OS rates among the 81 patients were 51.85% (42/81) and 12.35% (10/81), respectively. The 1-year and 5-year cumulative recurrence rates were 44.44% (36/81) and 100% (81/81), respectively. CD68 studies .Fig. 5SuWe also analyzed the prognostic roles of high expression of both CD68 and PD1 in ICC patients. Interestingly, ICC patients with high expression of both CD68 and PD1 showed a poorer prognosis compared to patients with high expression of only CD68 or PD1 and patients with low expression of both CD68 and PD1  Our results need to be validated in a randomized, controlled study using multiplex IHC assay. (2) The clinical efficacy of anti-CD47 in combination with anti-PD1 needs to be identified. (3) The complex immune profiles of ICC need to be fully uncovered.In conclusion, our study indicates that the phagocytosis checkpoints of the PD1/PDL1 axis and CD47/SIRPα are enhanced in the tumor tissues of ICC patients and especially in HBV infection patients. High expressions of PD1 or SIRPα in ICC patients predicts poor progression. Furthermore, anti-CD47 in combination with anti-PD1 may be a novel and promising treatment for ICC. Although correlation among the expression of CD68, SIRPα, and PD1 was revealed, further studies evaluating the other SIRPα-expressing or PD1-expressing cells will improve the understanding of the association between its expression and the tumor microenvironment in ICCs. Further studies are needed to confirm these findings.


Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineralization inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PPi hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hydrolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may exacerbate plaque calcification.(1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP Bglap) . Un. Uni), afication . Therefoormation ,19. Besiormation . As a refication . Under pfication ,23. Uponfication . Necrotification . ATP is fication ,27 and pfication  via ruptfication . Extracefication , ENPP1 [fication . TNAP [3fication  and CD39fication  dephosphfication  and 5′-efication . VSMCs efication ,33,34. Sic VSMCs . MOVAS pic VSMCs ,36,37.To monitor the transdifferentiation of MOVAS cells toward the osteochondrogenic phenotype, we determined the presence of calcium phosphate and its TNAP activity. The quantity of calcium relative to the mass of proteins increased from day 0 to day 21 A, which 31P-NMR and infrared spectroscopy were used to monitor the direct hydrolysis of ATP under physiological pH.TNAP activity is a marker for calcifying cells ,14. Take31P-NMR is a convenient method to quantify various nucleotides in a mixture  without isolation  or/and labeling [31P-NMR spectra from the whole reaction system, avoiding interference from intracellular nucleotides, nucleic acids, and phospholipids. MOVAS cells were incubated in osteogenic medium for 0, 7, 14, and 21 days. Then, after each incubation time in osteogenic medium, they were washed with Tris buffer (pH = 7.8), fixed with paraformaldehyde (PFA), and incubated in the presence of 50 mM extracellular ATP in Tris buffer with or without 5 mm Levamisole to determine the ATP hydrolysis rate. Extracellular media from fixed MOVAS cells were taken after 2, 24, 48, 72, and 96 h and analyzed by 31P-NMR. To illustrate the NMR findings, we selected those obtained on day 21 in the presence of 5 mM Levamisole , which correlated with a decrease in ATP, indicating slight hydrolysis of ATP (i (a singlet at +2.2 ppm). There was no formation of PPi (the singlet at −7.45 ppm was not observed) ruling out the hydrolysis of ATP into AMP and PPi. ATP hydrolysis increased from days 0 to 21 (p-nitrophenylphosphate (pNPP) as substrate. The addition of 5 mM levamisole, an inhibitor of TNAP, decreased ATP hydrolysis  decreased from 0 to 96 h, indicating significant hydrolysis of ATP . We obses of ATP . ADP was 0 to 21 . The pre 0 to 21 B, with pdrolysis . The hydobserved . This waobserved  allowingobserved .1 increased with the duration of incubation time from d9ay 0 to day 21, indicating a higher extracellular ATP hydrolysis rate, while k2 values, reflecting the extracellular ADP hydrolysis rate, were significant only from day 14 .The hydrolysis of PP−1), ADP (1212 cm−1), AMP (1088 cm−1), phosphate (1080 cm−1), and pyrophosphate (1107 cm−1) were identified from one of their respective phosphate vibration modes , −1 band corresponding to vibrational mode of phosphate belonging to ATP [−1 associated to the vibrational mode of phosphate [i , to ADP hydrolysis (i minus ADP), and to AMP hydrolysis  , as detedrocytes . That wake cells . Our finfication .31P-NMR data that more than two-thirds of the hydrolytic activity was inhibited in the presence of levamisole in fully trans-differentiated VSMC. This is consistent with previous reports that show that 75% of phosphatase activity in the plasma membrane is due to a levamisole-sensitive enzyme, probably TNAP [i in chondrocytes and osteoblasts [i from ATP, suggesting that the phosphodiesterase activity was very weak, while PPi is the major end product during hydrolysis of ATP by C6 rat glioma cells [i as compared to calcifying cells, which is coherent with the observed fivefold increase in PPi hydrolysis from non-calcifying MOVAS to calcifying MOVAS. This is also consistent with recent findings that report that pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis  was increased in both aortas and blood obtained from mice with Hutchinson–Gilford progeria syndrome [Extracellular nucleotides are released from the secretory pathway or via conductive/transport mechanisms, such as membrane connexin hemichannels, pannexin channels, and ATP conducting anion channels , or whenbly TNAP . Therefoeoblasts ,47,48. Hma cells . This insyndrome . Under psyndrome .pNPP . Phosphate buffered saline (PBS) pH 7.4 contained 137 mM sodium chloride, 10 mM phosphate, and 2.7 mM potassium chloride .Culture media, serum, streptomycin, penicillin Alizarin red (ARS), bicinchoninic acid, ® CRL-2797, Molsheim, FR) were cultured in Dulbecco’s Modified Eagle’s Medium rich in glucose (4.5 g.mL−1)  with L-glutamine (2 mM), streptomycin (0.1 mg.mL−1), penicillin (100 U), fetal bovine serum (10% v/v), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (20 µM). This medium is identified as the growth medium. Cultures were maintained in a humidified atmosphere consisting of 95% air and 5% CO2 at 37 °C. Cells were plated at a density of 10,000 cells.cm−2. Cells were grown confluent in growth medium supplemented with 50 μg.mL−1 ascorbic acid and 10 mM β-glycerophosphate , for 0, 7, 14, and 21 days . The medium was changed three times per week. These two osteogenic factors enable MOVAS cells to become calcifying vascular cells [Mouse MOVAS cells from ATCC , the extracellular medium was removed and MOVAS cells were washed with 1 mL of PBS. Deposited calcium was extracted from the extracellular matrix using 200 µL of 0.6 M HCl and kept overnight at room temperature. Then, 20 µL of the supernatant was mixed with 100 µL of 2-amino-2-methyl-1-propanol and 100 µL of a mix of 160 µm perature ,37. Abso® Infinite M200 Pro microtiterplate reader. Calibration was obtained using bovine serum albumin as standard.MOVAS cells grown at the indicated incubation times  were harvested in 200 µL of 0.1M NaOH and 0.1% SDS aqueous solution for calcium determination or in 400 µL Nonidet P-40 (0.2%) for the determination of enzymatic activity. Cells were disrupted by sonication then centrifuged 5 min (2000 g) at 4 °C. Then, 10 µL of supernatant was completed to 200 µL of reactive medium as reported ,37. It wp-NPP, 0.56 mM 2-amino-2-methyl-1-propanol and 1 mM MgCl2 in alkalinized distilled water (pH 10) was added [p-nitrophenolate (pNP) release was measured for 2 min every 10 s at 405 nm by TECAN® Infinite M200 Pro spectrophotometer. Specific activity was expressed as µmol of pNP released per minute and per milligram of protein (µmol.min−1.mg−1 or U.mg−1). Protein concentration was determined as indicated in MOVAS cells grown for the indicated incubation times  were harvested in 0.2% Nonidet P-40 and disrupted by sonication then centrifuged 5 min (2000 g) at 4 °C. Cell lysates were used to determine TNAP activity to demonstrate the VSMC transdifferentiation associated with increased TNAP expression, which is a marker for mineral competent cells ,35,36,42as added . The rat−1 ascorbic acid and 10 mM β-glycerophosphate, then they were washed with 1 mL of PBS, and fixed with 400 µL of 4% (v/v) PFA for 20 min at room temperature. PFA was then removed. PFA may affect the enzymatic activity of the cell surface. However, it was necessary to fix the cells with PFA since the incubation volume was too small to take aliquots of extracellular medium at time intervals. PFA or formalin were used to fix cells or tissues to determine activity or presence of ANKH [2, 5 µm ZnCl2) containing either 50 mM ATP or 50 mM PPi corresponding to a total of around 0.5 mL including cells. The reaction was performed with or without 5 mM Levamisole, a commercial inhibitor of TNAP. At the indicated time , 50 µL of reaction medium was sampled and stored at −20 °C prior to NMR or IR analyses.MOVAS cells were collected at the indicated time  in growth medium with 50 μg.mL of ANKH , ENPP [1 of ANKH , and TNA of ANKH ,42,58. T2H2O (10% (v/v) in Tris buffer) and 320 µL of Tris buffer . Auto-shim was done on 2H2O. 31P-NMR spectra were measured with a Ascend™ 600  or with a 300 ultrashield (Bruker®) spectrometer. Simultaneous ATP, ADP, AMP, PPi, and Pi peaks were monitored [31P. At least three independent measurements were performed to obtain kinetics parameters. In the absence of cells, the ATP hydrolysis was found to be negligible in the time course of the reaction.Herein, 40 µL of reaction medium see  was mixeonitored . Peaks w−1 resolution each and Fourier transformed. During data acquisition, the spectrometer was continuously purged with dry filtered air . For infrared measurements, an aliquot of 5 µL of reaction medium  flow-through cell (model TFC M13-3 Harrick Scientific Corp.). The phosphate band located at 1080 cm−1 has a molar absorption coefficient of 1215 m−1 cm−1 [IR spectra were acquired with a Thermo Scientific Nicolet iS10 spectrometer equipped with a DTGS detector. The IR spectra were recorded at 20 °C with 64 interferograms at 4 cmn medium  was depom−1 cm−1 . At leask1, k2, and k3 are the corresponding apparent first-order kinetic constants. The theoretical model was curve-fitted using the DYNAFIT software [31P-NMR and IR. At least three independent measurements were performed to evaluate errors. Assuming that the KM values of the phosphatases for ATP were below the mm range and because the substrate concentration was 50 mM, the rates could be assumed to be close to the Vm values. Therefore, the apparent first-order constants are strictly proportional to the phosphatase concentrations. To determine the ATP hydrolysis rate by the cells, Vm is expressed as nmol.min−1 using apparent first-order constant k1, neglecting variations of ADP and AMP from the 31P-NMR and IR spectra. Protein determination was performed according to The kinetic model of ATP hydrolysis assumes a sequential and irreversible mechanism described by Equations (1)–(3):software , using tp < 0.05 (*), highly significant when p < 0.01 (**), and extremely significant when p < 0.001 (***).For each analysis in i and not PPi is consistent with a possible molecular mechanism through which high ATP levels released by necrotic cells in atherosclerosis plaques generate Pi, exacerbating plaque calcification.The fact that MOVAS generated P