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Circulation, Volume 146, Issue Suppl_1, Page A10922-A10922, November 8, 2022. Introduction:Maternal hypercholesterolemia (MHC) during gestation is reported to enhance formation of fatty streaks in human fetuses leading to atherosclerosis in their adulthood. As maternal lipid levels increase during gestation as a normal physiological response to growing fetus, cholesterol levels are not routinely monitored in the outpatient clinic. The mechanisms mediating the stimulus transferred from hypercholesterolemic mothers to the fetus resulting in atherogenesis remains to be delineated.Hypothesis:We hypothesize that high lipid levels of hypercholesterolemic mother program the fetus by causing changes in DNA methylation patterns of lipid transport and metabolism genes in the placenta.Methods:Pregnant females within 100 days of first pregnancy were recruited. Total cholesterol (TC) and Low Density Lipoprotein (LDL) levels above 200mg/dL and 130mg/dL respectively were considered as hypercholesterolemic (MHC; n=50). Pregnant mothers with normal cholesterol levels were also recruited (NC; n=50). Lipid profiling of maternal blood was performed in all trimesters. Cord blood at the time of birth and neonatal blood on the 2ndday post birth were also profiled. DNA methylation array was performed in the placenta of MHC and NC mothers post delivery. Data were analyzed using RnBeads and Limma package. Data were validated by qRT PCR and immunohistochemistry of placental tissues.Results:Lipid profiling revealed high TC and LDL levels in MHC mothers and high triglycerides in their neonates on the 2ndday post birth. There were 3789 differentially methylated gene sets reported in the placenta of hypercholesterolemic mothers of which, 167 genes were hypomethylated and 94 genes were hypermethylated in the CpG islands. The hypermethylation and downregulation of lysosomal lipase genes (LIPA) was observed in the placenta of MHC mothers. This was complemented by upregulated expression of PEX11A, a peroxisomal biogenesis factor and ABCD1 which is a fatty acid transporter.Conclusions:We report the hypermethylation of lysosomal lipid degradation genes and hypomethylation of peroxisomal genes in the placenta of MHC mothers. This results in high triglycerides in neonates with a risk for early atherogenesis in adulthood.

Circulation, Volume 146, Issue Suppl_1, Page A14759-A14759, November 8, 2022. Introduction:Donor-derived cell-free DNA (%ddcfDNA) is a reliable non-invasive biomarker of acute rejection (AR). High-sensitivity cardiac troponin T (hs-cTnT) has been shown to be an easy to perform, low-cost, biomarker of myocyte injury.Hypothesis:We hypothesized that hs-cTnT correlates with %ddcfDNA in heart transplant patients and complements %ddcfDNA to improve the non-invasive diagnosis of AR.Methods:The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter, prospective, longitudinal cohort study of heart transplant patients transplanted between 2015 and 2021. The %ddcfDNA was quantitated with shotgun sequencing; hs-cTnT was measured on the Roche instrument. AR was ISHLT grade ≥2R acute cellular rejection (ACR) or pAMR ≥1 antibody-mediated rejection (AMR).Results:This analysis included 171 patients from the GRAfT cohort (mean age 52.3±12.4 yrs; 41% Black; 31% Female). Of the 1,030 simultaneous measurements of hs-cTnT and %ddcfDNA, 662 were paired with an endomyocardial biopsy. Median hs-cTnT was 1451 ng/L (IQR: 865-1931) on post-operative day 1 and decayed to 26 ng/L (IQR: 18-56) at approximately 90-days. The Spearman correlation between %ddcfDNA and hs-cTnT was ρ=0.52 (p

Circulation, Volume 146, Issue Suppl_1, Page A10202-A10202, November 8, 2022. Introduction:Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vascular obliteration of small pulmonary arteries (PAs), notably due to excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). Accumulating evidence indicates that PAH-PASMCs have acquired an over-efficient DNA damage response (DDR) ensuring sensing, signaling, and repair of DNA damages, accounting for their enhanced capacity to survive and proliferate under stressful conditions. DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery which plays an instrumental role in the overall survival and proliferation of cancer cells. Based on similarities between PAH and cancer cells, we thus hypothesized that increased expression of DNA-PK contributes to vascular remodeling in PAH.Methods and Results:Using Western blot (WB) and immunohistochemistry, we found that expression and activity of DNA-PK were increased in PAH-PASMCs (n=6) compared to controls (n=6, p

Circulation, Volume 146, Issue Suppl_1, Page A14938-A14938, November 8, 2022. Introduction:Pulmonary arterial hypertension (PAH) is a devastating disease characterized by obliterative vascular remodeling that leads to right heart failure and death. HIF-2α (hypoxia-inducible factor-2α) activation in lung endothelial cells is obligatory to obliterative pulmonary vascular remodeling in PAH patients and animal PAH models.Hypothesis:Endothelial cell-targeted nanoparticle can deliver plasmid DNA expressing the CRISPR/Cas9 system to disruptEpas1(encoding HIF-2α) selectively in endothelial cells in adult rats and inhibit PAH in monocrotaline rats.Methods:To target endothelium for robust genome editing, we developed poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide) (PEG-b-PLGA) copolymer-based nanoparticle formulated with polyethyleneimine. A single i.v. administration of mixture of nanoparticles and plasmid DNA expressing Cas9 controlled by ratCdh5promoter and guide RNAs targetingEpas1driven byU6promoter induced highly efficient genome editing in endothelial cells in 6-weeks old SD rats. RatCdh5-Cas9 plasmid DNA with scramble guide RNA were injected as a control group. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, hemodynamic measurement of right ventricular systolic pressure RVSP and weight ratio of right ventricular versus left ventricular plus septum (RV/LV+S), an indicator of RV hypertrophy were determined.Results:Single administration of mixture of nanoparticles and CRISPRCdh5plasmid DNA caused diminished HIF-2α protein expression selectively in endothelial cells indicating highly efficient genome editing. Genomic editing-mediated disruption ofEpas1in endothelial cells reduced RVSP and right ventricular hypertrophy as well as obliterative pulmonary vascular remodeling in MCT-induced PAH rats.Conclusions:These findings demonstrate robust genome editing ofEpas1via EC-targeted nanoparticle delivery of CRISPR-Cas9 plasmid DNA in adult rats leading to attenuated PAH in MCT rats, suggesting EC-targeted nanoparticle delivery of genome editing system to disrupt PAH-causing gene(s) in ECs is a promising precision gene therapy of PAH in patients.

Circulation, Volume 146, Issue Suppl_1, Page A9727-A9727, November 8, 2022. Introduction:BMPR2mutation is the most common genetic cause of pulmonary arterial hypertension (PAH). Loss ofBMPR2causes pulmonary arterial (PA) endothelial cell (EC) dysfunction and impairs EC-mediated suppression of smooth muscle cells proliferation. These are characteristic features of PAH vascular cells as is the presence of DNA damage that can be induced by oxidative stress. We previously showed that EC-Bmpr2-/-mice have persistent pulmonary hypertension (PH) with reoxygenation after hypoxia. However, the role of DNA damage in causing persistent PH related to loss of BMPR2 and oxidative stress is unknown.Hypothesis:We hypothesize that loss of BMPR2 causes unrepaired DNA damage and persistent PH by altering the expression of genes that maintain EC homeostasis.Methods and Results:DNA damage assessed by γH2AX foci was observed in PAEC of EC-Bmpr2-/-mice in room air, that was more extensive after reoxygenation (10% hypoxia for 3 weeks followed by recovery in room-air for 4 weeks). Deletion of the DNA damage sensor ataxia-telangiectasia mutated (ATM) in EC of mice (EC-Atm-/-) also resulted in persistent DNA damage and PH. RNA sequencing of PAEC after reoxygenation in EC-Bmpr2-/-and EC-Atm-/-mice vs. controls showed downregulated EC differentiation and development genes enriched in biding motifs forFoxf1, atranscription factor associated with DNA repair and angiogenesis.Foxf1expression was reduced in PAEC with loss of BMPR2 or ATM. Loss ofFoxf1decreased angiogenic genesCldn5andVegfr2, and DNA damage response genesAtmandp53, and impaired PAEC migration and tube formation.Foxf1overexpression increased these genes in PAEC. Immunohistochemistry showed decreased FOXF1 in PAEC of human pulmonary vascular lesions. Delivery ofFoxf1to EC-Bmpr2-/-mice with an AAV2-peptide targeting the PA endothelium attenuated DNA damage in PAEC, restored angiogenesis genes and completely reversed persistent PH returning right ventricular systolic pressure to normal levels.Conclusions:We link loss of BMPR2 and DNA damage sensing in PAEC to persistent DNA damage and PAH and show that both are mediated by reducedFOXF1. In EC-Bmpr2-/-mice,Foxf1gene therapy attenuated DNA damage restored angiogenesis genes, and prevented persistent PH.

Circulation, Volume 146, Issue Suppl_1, Page A9800-A9800, November 8, 2022. Introduction:Black heart transplant patients are at higher risk of acute rejection (AR) and death than White patients. Our group has shown that %donor-derived cell-free DNA (%ddcfDNA) is a sensitive and specific marker of AR in heart transplantation. Cell-free mitochondrial DNA (cfmtDNA) has been linked to allograft dysfunction and may mechanistically implicated in inferior outcomes for Black heart transplant recipients.Hypothesis:Higher levels of allograft injury when quantitated using %ddcfDNA and total cfmtDNA will be seen in Black heart transplant patients as compared to White patients.Methods:The Genomic Research Alliance for Transplantation (GRAfT) is a multicenter, prospective, longitudinal cohort study of heart transplant patients transplanted between 2015 and 2021. We included Black and White patients and shotgun sequencing was used to quantitate %ddcfDNA and polymerase chain reaction (PCR) to quantitate total cfmtDNA levels. AR was defined as grade ≥2R cellular rejection or pAMR ≥1 antibody-mediated rejection (AMR).Results:We included 151 patients (73 Blacks and 78 Whites), the median age was 56y (29% Female sex). Patients of both racial groups had comparable baseline characteristics. There were a ~1100 ddcfDNA and cfmtDNA measures included in the analysis. At 18-months, the incidence of AR was 2.8% in White patients and 9.2% in Black patients (p

Circulation, Volume 146, Issue Suppl_1, Page A10563-A10563, November 8, 2022. Introduction:Non-invasive quantification of donor-derived cell-free DNA (dd-cfDNA) has recently been shown to correlate with endomyocardial biopsy (EMB) for diagnosis of allograft rejection in heart transplant (HTx) patients. Hepatitis C virus-positive (HCV+) donor hearts are being more utilized with comparable survival outcomes.Hypothesis:The aim of this study is to evaluate the utility of dd-cfDNA monitoring in different types of rejections in recipients of HCV+ donor hearts.Methods:We performed a retrospective review of recipients of HCV+ donor hearts between 2018-2022 with paired dd-cfDNA/GEP and EMB results. The levels of dd-cfDNA/GEP were compared in different rejection types and no rejection for patients using non-parametric comparison. CMR ISHLT ≥ Grade 2R and AMR ≥ Grade 1 from histology were considered as allograft rejection.Results:There were 17 patients who underwent heart transplant from HCV+ donor heart. There was a total of 89 paired samples with dd-cfDNA and EMB. The reason for the biopsy was mostly for routine surveillance (N=68, 76.4%). There was a total of 1 case (1.1%) of grade 2 CMR and 16 cases of pAMR (18%; 10 cases of grade 1, 6 cases of grade 2). Levels of dd-cfDNA were elevated in cases of AMR when compared to no rejection (Median: 0.24% vs. 0.12%, respectively, P

Circulation, Volume 146, Issue Suppl_1, Page A9929-A9929, November 8, 2022. Background:Pulmonary arterial hypertension (PAH) is a lethal vasculopathy associated with pulmonary arteries remodeling and right ventricle (RV) dysfunction. Epigenetic dysregulation, including altered DNA methylation (DNAm), promotes PAH. However, the DNAm changes associated with PAH remain unexplored in human PAH RV and lungs. We conducted an exploratory study using human lung and RV samples to characterize the DNA methylome and transcriptomic changes associated with PAH.Methods/Results:We observed that PAH is associated with substantial changes in the DNAm landscape in both organs. We identified 88 identical differentially methylated probe (DMP) in both PAH Lungs and RV. Functionally, we observed that 65, and 76 of those DMP correlate with myocardial fibrosis and adverse pulmonary vascular remodeling, respectively. Then we clustered DMP onto functional differentially methylated region (DMR) and reported that 41% and 11.5% of the genes carrying a DMR are differentially expressed in PAH RV and lungs, respectively. Gene ontology analysis suggests that both impaired DNAm (DNA methylome) and genes expression (RNA sequencing) observed in PAH regulate biological functions related to inflammation, fibrosis, cell proliferation and vascular remodeling. Impaired DNAm landscape observed in PAH RV and lung is associated with the disease severity and contributes to 4.1% and 2.7% of the whole transcriptomic reprograming associated with PAH development in RV and lungs samples, respectively. Despite the modest overlap between DNAm and transcriptomic changes, we observed that DNAm affects genes and molecular pathways involved in PAH development.Conclusion:Although exploratory, our study is the first to characterize the DNAm and transcriptomic change associated with PAH in human RV and lung. Our data suggest that impaired DNAm landscape might contribute to the disease development/severity.

Circulation, Volume 146, Issue Suppl_1, Page A11956-A11956, November 8, 2022. BackgroundPrevious epigenetic studies in obesity interventions have focused on the intervention-associated DNA methylation (DNAm) changes, yet whether the DNAm before intervention could be used as a predictor of prognosis has not been investigated. Here we hypothesize that baseline DNAm can predict differential metabolic outcomes, i.e., changes in fasting plasma glucose (FPG) and glycated hemoglobin (HbA1c), induced by different weight-loss interventions.MethodsThe analyses included 75 individuals with obesity in the HeadsUp Study, a longitudinal study assessing the effectiveness of different weight-loss interventions, from the non-surgical intensive medical intervention (IMI), adjustable gastric band (BAND) and Roux-en-Y gastric bypass (RYGB) groups (n = 25 per group). Genome-wide DNAm was determined using baseline blood samples, while changes in FPG and HbA1c were measured at 1-year follow-up. Epigenome-wide association studies were performed on 455,983 CpGs to identify DNAm predictors of differential metabolic outcomes, adjusting for age, sex, BMI, FPG/HbA1c at baseline, weight loss and batch effect.ResultsDNAm levels at 3,216 and 117 CpG loci significantly predicted differential changes in FPG and HbA1c, respectively, when comparing RYGB vs. IMI groups, but not in other group comparisons (RYGB vs. BAND or BAND vs. IMI, with the exception of 6 CpGs identified for HbA1c changes when comparing RYGB vs. BAND), after FDR correction for multiple testing. Of these, altered DNAm at 79 CpGs significantly predicted changes in both FPG and HbA1c (RYGB vs. IMI). These CpGs were annotated to 68 genes, among which the most significant ones wereSNX18, LNPK, CCDC178, ADAM7andZNF23. These genes are mostly involved in adaptive thermogenesis, temperature homeostasis and regulation of cell population proliferation.ConclusionsTo our knowledge, this is the first study demonstrating that baseline blood DNAm predicts differential metabolic outcomes induced by different types of weight-loss interventions, independent of weight loss. Such results help explain, at least partly, the favorable metabolic outcomes of RYGB over other types of weight-loss intervention (e.g., BAND, IMI), and are likely to lead to precision strategies for obesity treatment.

Circulation, Volume 146, Issue Suppl_1, Page A11963-A11963, November 8, 2022. Introduction:Coffee intake has been associated with a lower risk of hypertension. Recent epigenome-wide association studies linked DNA methylation (DNAm) atPHGDH(cg14476101) with coffee consumption and blood pressure, which may explain the mechanism underlying coffee intake and blood pressure. However, little is known about whether the coffee-related DNAm is associated with long-term changes in blood pressure in response to dietary weight-loss interventions.Hypothesis:We hypothesized that participants with varying coffee-related DNAm levels may respond differently to dietary weight-loss interventions on long-term changes in blood pressure.Methods:The current study included 669 participants with overweight/obesity, who were randomly assigned to 1 of 4 diets varying in macronutrient components. Blood DNAm levels were profiled by a high-resolution methylC-capture sequencing at baseline. The regional DNAm atPHGDHwas defined as the average methylation level over CpGs within ±250 bp of cg14476101. Two-year changes in blood pressure were calculated. Linear regression models were used to examine the associations between DNAm atPHDGHand 2-year changes in blood pressure in response to dietary weight-loss interventions, adjusting for age, race, sex, body mass index, anti-hypertensive medications, and concurrent weight loss.Results:Regional DNAm atPHDGHwas not associated with 2-year changes in blood pressure in the total population. We found significant interactions between regional DNAm atPHDGHand dietary protein intake on 2-year changes in systolic blood pressure (SBP) and diastolic blood pressure (DBP) (p-interaction=0.018 and 0.005, respectively). Among participants assigned to an average-protein diet (15% of energy intake), higher baseline regional DNAm atPHDGHwas associated with greater reductions in SBP (beta [SE]: -9.7 [3.4], p=0.005) and DBP (beta [SE]: -6.5 [2.4], p=0.007); while no association was observed among those assigned to a high-protein diet (25% of energy intake; p=0.40 for SBP, p=0.15 for BP).Conclusion:Our data indicate that participants with a higher regional DNAm level atPHDGHbenefited more in long-term improvement in blood pressure when consuming an average-protein weight-loss diet.

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Ricerca per migliorare le cure nelle donne con cancro al seno 

Car-t e anticorpi bispecifici rivoluzionano la cura

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