Parp16 gene and increased H3K4me3 level to activate its host gene’s transcription. Intervention of either Smyd3 or PARP16 ameliorated vascular aging associated phenotypes in both cell and mice models. This study identified Smyd3-PARP16 as a novel signal axis in regulating UPR and endothelial senescence, and targeting this axis has implications in preventing vascular aging and related diseases.Vascular endothelial cell senescence and endoplasmic reticulum (ER) stress induced unfolded protein response (UPR) are two critical contributors to individual aging. However, whether these two biological events have crosstalk and are controlled by shared upstream regulators are largely unknown. Here, we found PARP16, a member of the Poly (ADP-ribose) polymerases family that tail-anchored ER transmembrane, was upregulated in angiotensin II (Ang II)-induced vascular aging and promoted UPR. Further, PARP16 was epigenetically upregulated by Smyd3, a histone H3 lysine 4 methyltransferase that bound to the promotor region of  Cellular senescence and unbalance of protein homeostasis, referred to as proteostasis, are two important hallmarks of aging and the key risk factor for age-related diseases such as cardiovascular disorder and cancer –3. SubseEndoplasmic reticulum (ER) is a central organelle involved in protein folding, maturation and quality control . In non-Vascular endothelial cell senescence is regarded as a response to endothelial cell dysfunction and contributes to the vascular diseases . ExtensiCdkn1a and leading to increased H3K4me3 level and gene expression. However, it remains unclear whether Smyd3 regulates Parp16 gene transcription, which affects ER stress and endothelial senescence.PARP16 , a member of the PARP/ARTD family, is a tail-anchored protein located at the endoplasmic reticulum membrane , and reqHere, we showed that PARP16 was upregulated and played a key role in promoting UPR and endothelial cell senescence. More importantly, PARP16 was epigenetically upregulated by Smyd3 and intervention of either Smyd3 or PARP16 could ameliorate vascular aging associated phenotypes in both cell and mice models. This study identified Smyd3-PARP16 as a novel signal axis in regulating UPR and endothelial senescence, and targeting this axis has implications in preventing vascular aging and related diseases.Atf4 (encoding ATF4 protein) and Hspa5 (encoding Bip protein) were significant upregulated while the activation of apoptotic gene Ddit3 (encoding CHOP protein) was modestly induced after Ang II treatment for 1 h  were also increased in Ang II induced VSMCs (Angiotensin II (Ang II), a major hormone that mediates atherosclerosis and hypertension , not onl for 1 h . We next for 1 h , suggest for 1 h . To conf for 1 h . Consisted VSMCs . These rIl-6 and Inos genes  or PARP16 short hairpin RNA (shPARP16) . The aorin vivo . After ain vivo . Furtherin vivo . We alsoin vivo , accompain vivo . These dReplicative senescence induces vascular cell growth arrest and loss of vascular homeostasis, contributing to the initiation and progression of cardiovascular diseases, we further examined the dynamic expression changes of PARP16 in replicative senescence of RAEC. Western blot showed an increase of PARP16 in RAECs undergoing replicative senescence . ER strePARP16, a tail-anchored protein located at the endoplasmic reticulum membrane, is required for activation of the functionally related ER stress sensors PERK and IRE1 during the UPR. Here we have identified that PARP16 promotes vascular cell senescence and ER stress, and blocking PARP16 significantly alleviated the senescence-associated phenotypes in Ang II-treated mice and vascular cell. Further, PARP16 was epigenetically upregulated by Smyd3, a histone H3 lysine 4 methyltransferase that bound to promotor of PARP16 and increased H3K4me3 level to activate its host gene’s transcription. Finally, Smyd3-PARP16 signal axis mediated senescence and ER stress exist in cell models (Ang II-induced premature aging and replicative senescence), animal models (Ang II-infused mouse).PUMA, p53, NOXA, Bax and cell viability were not changed obviously after Ang II treatment (data not shown). Further, PARP16, a tail-anchored ER transmembrane protein required for activation of the functionally related ER stress. PARP16 has been reported as an effective activator of ER stress sensor PERK and IRE1α through increasing their kinase activities and endonuclease activity of IRE1α. In this study, we have demonstrated PARP16 is involved in Ang II-induced vascular senescence. On contrary, knocking down of PARP16 led to reversed senescence-related phenotypes including cell-cycle arrest and β-Gal staining, at least partly through mediating PERK and IRE1α branches of ER stress. EGCG has been reported is a potential inhibitor of PARP16, which suppressed the ER stress-induced p-PERK and the transcription of UPR-related genes. We also showed that EGCG reversed senescence-related phenotypes and raised blood pressure caused by Ang II. These results indicated that agents targeting PARP16, such as EGCG or gene intervention probably provide new ideas for clinic prevention of age-related cardiovascular diseases.Aging is one of the causes of various cardiovascular diseases and perturbs a number of metabolic and hemodynamic mechanisms in the cardiovascular system in general and the vascular endothelium in particular , 27. ProParp16’s promotor regions, and transcriptionally activated PARP16 to promote the appearance of aging phenotype, providing a previous unestablished association between Smyd3 and PARP16.In our previous study, we found Smyd3, an epigenetic writer, was increased in vascular ageing. The expression of Smyd3 was up-regulated in Ang II-induced vascular senescence and knocking down of Smyd3 reversed senescence-related phenotypes and elevated blood pressure. In this study, we further found knocking down of Smyd3 significantly depleted PARP16 expression along with the reduced expression of ER stress and senescence-related markers, which stated that Smyd3 knockdown may inhibit ER stress through suppressing PARP16 expression. Besides, the consistent results could be drawn from the application of Smyd3 specific inhibitor. Previous studies indicated that Smyd3 was elevated in cancer-associated phenotypes through methylation of histones  and non-histone proteins . In addiThe activation of the UPR seems to occur in all types of senescence, whether the inducer is successive replications , oncogenParp16 and increased H3K4me3 level and ultimately activated its transcription. To the best of our knowledge, we discovered Smyd3-PARP16 signal axis as a novel and important factor in regulating Ang II-induced endothelial senescence and function disorder. Such mechanism may also exist in replicative senescence model and also in Ang II infusion mice model , epigallocatechin gallate , 4μ8C (a specific IRE1 inhibitor) and ISRIB (a specific PERK inhibitor) were purchased from Meilunbio . EPZ031686 was purchased from MCE . Antibodies were obtained from the following commercial sources: PARP16, Smyd3, H3K4me3, Bip and XBP-1 were purchased from Abcam; VCAM-1, Calnexin, and GAPDH was purchased from Proteintech ; IL-6 and iNOS were purchased from Epitomics ; p-PERK, p-eIF2α, p53, p21 and p16 were purchased from Cell Signaling Biotechnology ; p-IRE1α were purchased from Thermo Fisher ; ATF6 was purchased from Bioss .via their tail veins after, respectively. In total, 4 injections were administered, once every 5 days, each injection contained 0.15 mL of the concentrated viral suspension with a titer of 1×108 IU/mL.C57BL/6 mice were randomly assigned to one of three groups: The sham control group, Ang II-infusion group or shRNA PARP16 group (KD PARP16). Mice were anesthetized with isoflurane, followed by subcutaneous implantation of an Alzet osmotic minipump  containing only saline, or Ang II  dissolved in saline. Mice were then continuously infused with saline or Ang II (1.5 mg/kg/day) for 4 weeks. Infusion 5 days later, KD PARP16 group and Ang II-infusion group were injected with lentiviral vector expressing shRNA PARP16 and shRNA scramble (sh MOCK) For EGCG groups, after mice were subcutaneously implanted with osmotic minipump, EGCG at doses of 20 and 50 mg/kg/day was administered to mice by intraperitoneal injection for 4 weeks.C57BL/6 mice were sacrificed by cervical dislocation and the thoracic aorta of each mouse was cut into two sections. One section was stored at -80°C for protein extraction. The other section was fixed with 4% paraformaldehyde overnight, and was then embedded in paraffin for immunofluorescence staining. Paraffin-embedded sections (8 μm thick) were cut every 200 μm length of thoracic aortas from the proximal thoracic aortas specimens.RAECs were obtained as our described . Cells tFor the replicative senescence, the cells then underwent serial passaging to reach senescence. The senescent status was verified by staining for SA-β-gal. 90% percent of the cells at 10 passages stained positive for SA-β-gal.Parp16 gene were as follows: mouse_Parp16_shRNA1_F: 5'-CCGGCAAGTGCCAAATCAAGAAGAACTCGAGTTCTTCTTGATTTGGCACTTGTTTTTG-3'; mouse_Parp16_shRNA1_R: 5'-AATTCAAAAACAAGTGCCAAATCAAGAAGAACTCGAGTTCTTCTTGATTTGGCACTTG-3'; mouse_Parp16_shRNA2_F: 5'-CCGGCCTGAACAAGACTTCTCTGTTCTCGAGAACAGAGAAGTCTTGTTCAGGTTTTTG-3'; mouse_Parp16_shRNA2_R: 5'-AATTCAAAAACCTGAACAAGACTTCTCTGTTCTCGAGAACAGAGAAGTCTTGTTCAGG-3'. The oligos contain the shRNA sequence flanked by sequences that are compatible with the sticky ends of EcoR I (NEB) and Age I (NEB). Forward and reverse oligos are annealed. Then pLKO.1 TRC Cloning Vector (Addgene) was digested with restriction endonucleases EcoR I and Age I. After that, the PARP16 insert and linearized plasmid were joined by T4-DNA ligase (NEB) and ligated into the pLKO.1 TRC Cloning Vector, producing a final plasmid that expresses the PARP16 shRNA.The sequences of shRNA targeting mouse For PARP16 overexpression (PARP16 OE) and Smyd3 overexpression (Smyd3 OE) plasmid construction, the rat genomic DNA was used to amplify the PARP16 and Smyd3 full-length cDNA, then cloned into the vector pCDH-CMV-MCS-EF1-copGFP. The product was confirmed by sanger sequencing.via ultracentrifugation for 2 h at 250,000 rpm. The viral particles were resuspended in PBS and stored at -80°C. Lentivirus encoding PARP16, Smyd3 or control vectors in the presence of 8 μg/ml polybrene (Sigma) was added to RAECs culture media for 24 h. This medium was then removed and the cells were allowed to incubate for additional 48 h under normal culture conditions.To obtain the lentivirus, the recombinant plasmid and packaging vector psPAX2 and PMD2.G were co-transfected into 293T cells using transfection reagent lipofectamine 2000 . After incubation for 48 h, the lentivirus in the culture medium was collected by filtration with 0.45 μm filters and then concentrated Rat PARP16, Smyd3, and control siRNA were produced by GenePharma . The sequence of the siRNA was as follows: PARP16: 5’-CCUACCUCACAAGUGACUUTT-3’; and 5’-AAGUCACUUGUGAGGUAGGTT-3’. Smyd3: 5'-CUGAUGCGGUGUUCUCAAUTT-3', and 5'-AUUGAGAACACCGCAUCAGTT-3'. Negative control: 5’-UUCUCCGAACGUGUCACGUTT-3’. To introduce siRNA into RAECs, the cells were plated on 6-well plates at 30% to 50% confluence before transfection. Individual siRNAs (15 nM), Lipofectamine RNAiMAX, and Opti-MEM were mixed and incubated at room temperature for 5 min. siRNA-lipofectamine RNAiMAX complexes were added to cells and incubated for 24 h, then the medium was replaced with fresh serum DMEM medium. Experiments were performed 72 h after transfection.To assess cell proliferation, immunofluorescence assay was also used for the detection of EdU incorporated into cellular DNA . The immunofluorescence detection was performed according to the manufacturer's instructions. Total cellular nuclei were stained with DAPI. Immunofluorescence EdU positive cells were observed under the Zeiss inverted fluorescent microscope. And the EdU incorporation rate was expressed as the ratio of EdU positive cells to total DAPI positive cells (blue cells).The expression of senescence-associated β-galactosidase  in cells was determined by SA-β-Gal staining as described . SA-β-GaTM Real-Time PCR System . Primer sequences of genes are listed in the −ΔΔCt equation, with GAPDH as control for normalization.Total RNA was extracted from RAECs with TRIzol Reagent  following the manufacturer’s instructions. Total RNA (600 ng) of each sample was reversely transcribed into cDNA using two-step RT Kit  according to the manufacturer’s directions and Real-time qPCR was performed with Bio-Rad CFX ConnectPolyA+ RNA was enriched by oligo(dT)25 Dynabeads (Invitrogen) from total RNA extracted as above. The dUTP-based strand-specific RNA-seq libraries for control and Ang II induced RAEC cells were constructed according to the protocol of Parkhomchuk et al. . ChIP-seParp16 gene promoter subjected to PCR analysis. Primer sequences are as follows: Parp16: 5’-CAGGACTGACTGCAGAGTGC-3’, and 5’-ACTCTGTAGCCCATGCTGAC-3’. Thermal cycling was carried out as follows: 94°C for 30 s; 30~40 cycles of 94°C for 30 s, 52~58°C for 30 s and 72°C for 30 s; 72°C for 10 min; hold at 4°C. The PCR product was visualized in a 2% agarose gel stained by Gel-Red.Both H3K4me3  and Smyd3 antibody  were used during the immunoprecipitation step before PCR. Taq DNA Polymerase (Yeasen) was used for the PCR step, and the multiple sets of primers spanning the transcription factor binding site on Samples were prepared with RIPA buffer  containing protease and phosphatase inhibitor cocktail . Whole lysates samples were separated by SDS-PAGE and blotted to nitrocellulose membrane. Protein bands were detected with fluorophore-conjugated secondary antibodies, and detection and analysis were performed with the Odyssey imaging system (LI-COR).Cells were seeded on glass coverslips placed in 24-well plates. Cells were fixed with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.25% Triton X-100 in PBS 10 min. Next, the slides were blocked in PBS with 10% goat serum for 30 min, and incubated overnight with primary antibodies at 4°C. Appropriate secondary antibodies were added and incubated with cells for 1.5 h at room temperature. The nuclei were stained with DAPI. The images were captured by using a fluorescence microscope .p values less than 0.05 were considered significant.Statistical analysis was performed using the software GraphPad Prism version 7.0. All values are expressed as the mean ± S.D. One-way ANOVA were initially performed to determine whether an overall statistically significant change existed before using the two-tailed paired or unpaired Student’s t-test. For each test, Male C57BL/6 mice (22-25 g) were purchased from SHANGHAI SLAC LABORATORY ANIMAL CO. LTD . All animals were housed under conventional conditions in the animal care facilities and received humane care in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals. The experimental protocol conformed to the Animal Welfare Act Guide for Use and Care of Laboratory Animals, and was approved by Institutional Animal Care and Use Committee (IACUC), School of Pharmacy, Fudan University, China.Supplementary FiguresSupplementary Table 1


To compare different β-amyloid (Aβ), tau, and neurodegeneration (AT[N]) variants within the Swedish BioFINDER studies.42 and amyloid-PET ([18F]flutemetamol); for T, CSF phosphorylated tau (p-tau) and tau PET ([18F]flortaucipir); and for (N), hippocampal volume, temporal cortical thickness, and CSF neurofilament light (NfL). Binarization of biomarkers was achieved using cutoffs defined in other cohorts. The relationship between different AT(N) combinations and cognitive trajectories  was examined using linear mixed modeling and coefficient of variation.A total of 490 participants were classified into AT(N) groups. These include 53 cognitively unimpaired (CU) and 48 cognitively impaired (CI) participants (14 mild cognitive impairment [MCI] and 34 Alzheimer disease [AD] dementia) from BioFINDER-1 and 389 participants from BioFINDER-2 (245 CU and 144 CI [138 MCI and 6 AD dementia]). Biomarkers for A were CSF AβAmong CU participants, A−T−(N)− or A+T−(N)− variants were most common. However, more T+ cases were seen using p-tau than tau PET. Among CI participants, A+T+(N)+ was more common; however, more (N)+ cases were seen for MRI measures relative to CSF NfL. Tau PET best predicted longitudinal cognitive decline in CI and p-tau in CU participants. Among CI participants, continuous T  and (N) measures improved the prediction of cognitive decline compared to binary measures.Our findings show that different AT(N) variants are not interchangeable, and that optimal variants differ by clinical stage. In some cases, dichotomizing biomarkers may result in loss of important prognostic information. The National Institute on Aging and Alzheimer's Association has proposed a framework for research2 based on the idea that biological processes start before symptoms in AD. The framework suggests that biomarkers of Aβ (A), tau (T), and neurodegeneration (N) can be compiled into a classification system (AT[N]). The framework is purposely agnostic to the details of operationalization, and it is unknown how different variants of AT(N) (using different biomarkers) compare. We therefore tested a variety of AT(N) approaches, with different combinations of biomarkers, which were compared in terms of prevalence in cognitively unimpaired (CU) and cognitively impaired (CI) participants and to predict cognitive decline. We hypothesized that different AT(N) variants would have considerable differences in category prevalence and that the prediction of longitudinal cognitive decline would vary both by AT(N) variant and predictor data type (binary vs continuous).Alzheimer disease (AD) is characterized by accumulation of β-amyloid (Aβ) and tau, atrophy, and cognitive decline.2–4 Exclusion criteria included presence of objective cognitive impairment, severe somatic disease, and current alcohol or substance abuse. Patients with MCI fulfilled the DSM-5 criteria for minor cognitive impairment.5 Patients with AD dementia fulfilled the DSM-5 criteria for major cognitive impairment due to AD.5 Patients with MCI and patients with AD had low CSF Aβ42/Aβ40 levels (<0.10). Exclusion criteria were cognitive impairment that could better be accounted for by another non-neurodegenerative condition, severe somatic disease, and current alcohol or substance abuse. Longitudinal cognitive data were available in all individuals from BioFINDER-1. To validate cross-sectional results, we also included 389 participants from the Swedish BioFINDER-2 study (245 CU and 144 CI participants) .We included individuals from the Swedish BioFINDER studies . BioFINDER-1 included 53 CU and 48 CI participants (14 mild cognitive impairment [MCI] and 34 AD dementia). CU individuals were aged ≥60 years and did not have MCI or dementia.All participants gave written informed consent. Ethical approval was given by the Regional Ethical Committee of Lund University. Approval for PET imaging was obtained from the Swedish Medical Products Agency and the local Radiation Safety Committee at Skåne University Hospital.6 CSF Aβ (Aβ42), tau phosphorylated at Thr181 (p-tau), and neurofilament light (NfL) were quantified using ELISAs .7 CSF analyses were performed in accordance with the Alzheimer's Association Flow Chart for CSF biomarkers.8Procedures for CSF collection, processing, and storage have been described.9 FreeSurfer  was used to extract hippocampal volume  and a cortical thickness measure within a meta–region of interest (ROI) encompassing temporal regions with known susceptibility in AD .10T1-weighted MRI was performed on a 3T Siemens Tim Trio scanner  using a sagittal magnetization-prepared rapid gradient echo sequence.18F]flutemetamol and [18F]flortaucipir PET, respectively, as described elsewhere.12 Briefly, dynamic (list-mode) studies were performed over the 90- to 100-minute postinjection interval for amyloid PET and 80- to 100-minute interval for tau PET. In BioFINDER-2, tau PET was performed using [18F]RO948,13 with dynamic (list-mode) studies performed over the 70- to 90-minute interval.Amyloid and tau imaging were performed using flutemetamol and the inferior cerebellar cortex for [18F]flortaucipir and [18F]RO948.Target ROIs were selected on the basis of previously published findings: a global neocortical composite for amyloid PETGlobal cognition was assessed using longitudinal Mini-Mental State Examination (MMSE) (tested at an average [median] of 4 time points [interquartile range (IQR) 3–5] over a median time span of 4.7 years [IQR 8 months to 4.2 years]).42 (A1), amyloid PET  (A2), CSF p-tau (T1), tau PET ([18F]flortaucipir) SUVR within the ITC (T2) and composite Braak V/VI region (T3), hippocampal volume (N1), cortical thickness within the temporal meta-ROI (N2), and CSF NfL (N3). Binarization of biomarkers  was done using cutoffs established using Gaussian mixture modeling (GMM) in BioFINDER-1 for amyloid PET, CSF, and MRI-based measures, and in Alzheimer's Disease Neuroimaging Initiative (ADNI) for tau PET . The only exception was hippocampal volume, which was not suitable for GMM due to a clear unimodal distribution. We therefore used a Youden index–derived cutoff instead . This method, along with the mean ±2 SD from Aβ-negative CU controls , were also used for all biomarkers as a sensitivity analysis to examine the effect of cutoff selection.AT(N) biomarkers included CSF Aβ42 is the A biomarker in the variant A1T1[N]2).These 4 terms are used throughout the article: (1) “AT(N) variant,” a specific combination of A, T, and (N) 2 is a variant); (2) “AT(N) category,” a combined biomarker profile in an AT(N) variant ; (3) “AT(N) component,” refers to the A, T, or (N) position; and (4) “AT(N) biomarker,” refers to a specific measure for a component , with significance set at p < 0.05, 2-tailed.Demographics and biomarkers were compared between diagnostic groups using Kruskal-Wallis test or Fisher exact test. Associations between continuous biomarkers were summarized using Spearman rank correlation (ρ), Cohen kappa statistic (κ), and percentage agreement (concordance). Prevalence estimates for AT(N) categories were calculated in CU and CI participants with confidence intervals generated using bootstrap resampling . Prevalence findings for AT(N) categories were further examined in a separate cohort (the Swedish BioFINDER-2 study). The relationship between AT(N) categories and cognitive trajectories  was examined using linear mixed-effects (LME) modeling, with subject-specific intercepts and slopes, adjusted for age, sex, and education, with a restricted cubic spline term for time, to account for nonlinear trajectories. LME models with different AT(N) variants as predictors were compared by marginal Anonymized study data for the primary analyses presented in this report are available on request from any qualified investigator for purposes of replicating the results.APOE ε4. There was a trend that CU participants were older in BioFINDER-1, and they were significantly younger than CI in BioFINDER-2. CI participants had lower MMSE, Aβ42, hippocampal volume, and temporal meta-ROI cortical thickness, and higher p-tau, amyloid and tau PET SUVRs, and NfL. As CSF NfL has been shown to be associated with age16 we divided participants into younger and older using a median split and compared CSF NfL levels between resulting groups. We found that there was no significant difference in NfL levels between groups (p = 0.365); this was also the case when dividing the participants using age 65 as a cutpoint (p = 0.590), and when running the same analyses in BioFINDER-2. Participant numbers and median NfL values for these 2 comparisons are summarized in table e-4 (doi.org/10.5061/dryad.p5hqbzkkx). On the basis of these analyses, the prevalence of (N)+ using NfL is unlikely to vary by age in the present cohort.Demographics are presented in 42 < 510.4 ng/L (A1), amyloid PET >0.743 SUVR (A2), p-tau >60.2 ng/L (T1), ITC tau PET >1.29 SUVR (T2), Braak V/VI tau PET >1.32 SUVR (T3), hippocampal volume  <0.00215 (N1), temporal meta-ROI thickness <2.38 mm (N2), and CSF NfL >1850 ng/L (N3). Similar cutoffs were obtained using the Youden index and mean ± 2 SD from Aβ-negative CU controls, except for CSF Aβ42, where the mean −2 SD resulted in a more conservative cutoff .Cutoffs were defined as CSF Aβ42 (A1) vs amyloid PET (A2) , between p-tau and tau PET SUVR in the ITC  and Braak V/VI meta-ROI , and between the 2 tau PET measures . For measures of neurodegeneration, the imaging measures had slightly better concordance with each other  than with NfL .doi.org/10.5061/dryad.p5hqbzkkx). When examining A and T only among CU participants (doi.org/10.5061/dryad.p5hqbzkkx), most were negative for both when using amyloid PET (A2), with a higher percentage showing isolated amyloid positivity when using CSF Aβ42 (A1). Positivity in both A and T was highest when using CSF p-tau (T1) . The prevalence of T+ was much less when using tau PET SUVR in ITC (T2) (9% were T+ in the A1+ and A2+ groups) or tau PET Braak V/VI SUVR (T3) (0% T+). These results show that CSF p-tau (T1) results in a much higher prevalence of T+ compared to tau PET in CU participants. Similar findings  were found using Youden and mean ±2 SD based cutoffs  and using GMM in the BioFINDER-2 cohort (doi.org/10.5061/dryad.p5hqbzkkx). The pattern for tau biomarkers also held in both cohorts when using tau PET in a very early ROI .Prevalences for AT(N) categories based on GMM cutoffs are summarized for CU and CI participants in doi.org/10.5061/dryad.p5hqbzkkx). Although all 8 possible categories were represented in some AT(N) variants, several categories were absent or had very low frequencies . Among the different biomarkers for (N), hippocampal volume (N)1 resulted in more (N)+ cases than when using cortical thickness in the temporal lobe meta-ROI ([N]2) and CSF NfL ([N]3) (doi.org/10.5061/dryad.p5hqbzkkx) and in the BioFINDER-2 cohort (doi.org/10.5061/dryad.p5hqbzkkx). Overall, however, fewer A+ cases were seen among CU participants in BioFINDER-2 as compared to BioFINDER-1 .When adding (N) biomarkers , A−T−(N)L ([N]3) . This fidoi.org/10.5061/dryad.p5hqbzkkx). However, when using the Braak V/VI ROI for tau PET (T3), A+T−(N)− was seen in approximately half of participants, because T3+ was relatively rare. Using A, T, and (N) components (doi.org/10.5061/dryad.p5hqbzkkx). Similar findings were observed when using Youden and mean ± 2 SD based cutoffs . A comparable pattern of prevalence findings was also seen for CI participants in BioFINDER-2  and temporal cortex thickness ([N]2) as compared to CSF NfL ([N]3) (doi.org/10.5061/dryad.p5hqbzkkx). The prevalence of the A+T+(N)+ category was lower across AT(N) variants in BioFINDER-2 as compared to BioFINDER-1 .Positivity for both A and T components was the main finding when using only biomarkers for A and T in CI doi.org/10.5061/dryad.p5hqbzkkx). Using continuous predictors, the AT(N) variant combining amyloid PET, p-tau, and temporal cortical thickness (A2T1[N]2) best captured changes in cognition in CU participants (R2 = 0.339)  showed that all included variables  contributed significantly to the prediction of cognitive decline. The 2 best models in CU included amyloid PET (A2) and temporal meta-ROI cortical thickness (N)2 (R2 = 0.554)  again showed that all 3 biomarkers contributed significantly. Among CI, the 2 models that best predicted cognitive decline included amyloid PET (A2) and tau PET ITC SUVR (T2)  . Compariess (N)2 . By cont= 0.554) . In thisUVR (T2) .R2 for CU (0.292) and CI (0.463) participants, respectively. Overlap among the best 5 models from continuous and binary data was partial ; the range of R2 values across models was narrow for CI participants (range 0.357–0.463) and somewhat broader for CU participants (range 0.090–0.292). Among the top 5 best models, the shift from continuous to binary data resulted in an average decrease in R2 of 0.101 for CU participants and 0.111 for CI participants.We next tested the effect of switching from continuous to binary data for the biomarkers. This led to A2T2(N)2 and A2T2(N)3 having the highest R2 from continuous data) models for MMSE in CU and CI. Findings are presented in doi.org/10.5061/dryad.p5hqbzkkx) (models 2–5). Among CU participants, the differences in R2 between binary and continuous predictors were small for CSF Aβ42 (A1)  but greater for amyloid PET (A2) . A larger difference in R2 was seen between binary and continuous data for p-tau (T1)  and CSF NfL ([N]3) , but not for tau PET or MRI measures of neurodegeneration in CU . Among CI participants, little difference in R2 was observed for binary and continuous A biomarkers, suggesting that the dichotomization captured most relevant variability in the A biomarkers . However, higher R2 values were consistently seen when using continuous measures of T  and (N) , suggesting that valuable information was lost when dichotomizing T and (N) biomarkers in CI. To explore alternatives to binary vs continuous biomarker groups, a further analysis was performed combining binary and quartile predictors. This confirmed the patterns observed when combining binary and continuous data (doi.org/10.5061/dryad.p5hqbzkkx).Due to the observed differences between continuous and binary data, we tested mixing binary and continuous predictors. To reduce the number of comparisons, we performed these analyses only on the top 5 best  variants confirmed our hypotheses that different operationalizations of the AT(N) system have strong effects on category prevalence and predictions of future cognitive decline. Our first main finding was that different AT(N) variants, using different biomarkers for A, T, and (N), give considerable differences in the classification of CU and CI participants. For example, a much larger proportion of CU participants are classified as T+ using CSF p-tau than when using tau PET, and a larger proportion of CI participants are classified as (N)+ using MRI measures than when using CSF NfL. The second main finding was that different AT(N) variants have different associations with longitudinal cognition, with differences between CU and CI . The third main finding was that using binary (rather than continuous) biomarker data affects prediction of longitudinal cognition, and some biomarkers were more suitable than others for dichotomization. Taken together, these results show that different AT(N) variants result in both different classifications of individuals at baseline and differences in the prognosis of future cognitive decline. This has implications for how to use and interpret AT(N) in research studies, clinical trial design, and potentially also in clinical practice. For example, prevention trials focusing on the very early  stages of AD may benefit from using CSF p-tau to define T+, but prevention trials in the clinical stage  may benefit from using tau PET instead.17 and our findings show that these modalities are not always interchangeable, especially not in all clinical disease stages. This is in line with a recent study that found that concordance between AT(N) biomarkers varied across CU and CI groups, and appeared to be stage dependent.18 Although the overall prevalence of amyloid positivity among CU individuals has been shown to be similar between cohorts assessed with CSF Aβ42 or amyloid PET,19 our finding (from a direct comparison within the same participants) that the proportion of CU participants defined as A+ was higher when using CSF Aβ42 suggests that amyloid imaging alone may underestimate early amyloid positivity. This is in line with previous findings in other cohorts that CSF Aβ42 may capture early stage amyloid pathology before amyloid PET.21 Similar to A, tau positivity was much higher among CU participants using p-tau, and a greater difference was seen between CSF p-tau and tau PET. This may be due to a temporal lag between these measures or differences in variance in both the negative range (likely greater for CSF) and the positive range (likely greater for PET).22 The discrepancy between prevalence when using NfL and MRI-based measures of (N) was a somewhat unexpected finding, given that both CSF NfL and MRI-based measures are altered in MCI and AD.24 However, following initial increases, the change in NfL may slow during the symptomatic course of the disease.26 Group differences in NfL (CI > CU), combined with similar levels across MCI and AD and the fact that NfL was only a significant predictor of longitudinal MMSE among CU participants, supports this scenario.The AT(N) system includes both fluid and imaging biomarkers,In order to verify the prevalence findings across AT(N) categories, we repeated prevalence calculations in a separate and larger cohort (the BioFINDER-2 cohort). Similar to findings in BioFINDER-1, we found higher prevalence of T+ using CSF p-tau and (N)+ using hippocampal volume among CU participants. Further, we found fewer T+ cases using tau PET in the Braak V/VI region and more (N)+ cases using MRI measures, as compared to CSF NfL, among CI participants. These findings were validated despite the fact that the BioFINDER-2 cohort used other technologies for tau PET and CSF biomarkers than BioFINDER-1. These findings therefore strongly support the idea that biomarker interchangeability across A, T, and (N) categories is likely to vary according to biomarker selection and clinical stage of the disease. Some differences were observed between cohorts, however, in terms of the overall prevalence of certain AT(N) variants, due to the BioFINDER-2 cohort having an overall lower frequency of A+ participants and a CI group comprising very few dementia cases as a result of the study having only recently been launched. Finally, while the cutoffs identified using the Youden index and mean ± 2 SD from Aβ-negative controls as part of the sensitivity analysis were similar to those using GMM, some differences were noted for A1 and (N)3. Though the 3 methods used are well-established, this finding highlights the optimization of cutoff selection  as an important area for future studies.18F]flutemetamol as the A component, consistent with findings that amyloid PET is a later marker than CSF Aβ42.20 In line with findings that p-tau behaves as an early disease state marker in AD27 , while tau PET behaves more as a stage marker ,28 the best model for predicting cognition in CU (A2T1[N]2) included p-tau as the T variable, while tau PET was clearly superior in CI individuals. This is consistent with a temporal ordering of tau biomarkers where p-tau is most dynamic during the preclinical phase of AD, and tau PET in the clinical phase. Even among CU individuals, however, tau PET was associated with cognitive decline. Though few CU participants had elevated tau PET values, half had ITC SUVR values 20%–30% above the reference region, in line with the known association between cognition and tau PET signal within this region.14 The distribution of (N) biomarkers across the best models  may reflect variability in the patterns of neurodegeneration biomarkers across the AD continuum,29 with CSF NfL being most dynamic during the preclinical disease phase,30 similar to CSF Aβ42 and p-tau.Most AT(N) variants with stronger associations with longitudinal cognition had [R2 values decreased when binarizing amyloid PET, CSF p-tau, and NfL, but were relatively unaffected when tau PET and MRI-based measures were dichotomized. By contrast, dichotomization of tau PET and MRI-based measures decreased R2 values among CI participants. Though the use of quartiles was superior to binary data, our findings do not support them being an optimal alternative to continuous data. These findings require confirmation in follow-up studies with larger cohorts and more varied cognitive measures, but they point to the fact the binarization may lead to loss of information when the tested biomarker changes on a continuum  rather than more clear transitions from one stage to another . In practice, this means that more complex models that take continuous data into account may outperform simple binarization, for example when selecting participants likely to decline cognitively in a clinical trial.In line with our hypotheses, the observed differences between models with binary or continuous biomarkers indicate that dichotomization in some cases may decrease sensitivity to predict changes in cognition. Moreover, permutation findings suggest that biomarkers that are dynamic at a given disease stage are less suited to binarization; among CU participants, for instance, 31–33 Further, the use of the Braak V/VI ROI, reflecting widespread neocortical tau,34 was of greater relevance to the CI group, as neocortical tau pathology is almost invariably associated with cognitive impairment.35 Findings among CU participants, however, did not differ when using a region important for very early tau pathology 34 2 remained the best predictor of longitudinal cognition). Finally, although this is, to our knowledge, the most comprehensive study to date testing different biomarkers for AT(N), we acknowledge that several other biomarkers could also be tested .37The study has limitations. First, the sample size for the analysis of longitudinal cognition was modest, which may reduce the power to detect associations between cognition and certain AT(N) variants. The use of the MMSE may have precluded insight into domain-specific cognitive decline, including nonamnestic aspects, as well as neurobehavioral changes that may be the first symptom of AD pathophysiology during the preclinical phase of AD.38 The large variability in how participants are classified when using different biomarkers shows that different AT(N) variants are not interchangeable. Optimal biomarker combinations for diagnosis and prediction of rapid cognitive decline may vary by clinical stage. Moreover, dichotomizing some biomarkers results in the loss of important information compared to using them on continuous scales.The AT(N) system is useful to classify participants, predict cognitive decline over time, and perhaps stratify participants for inclusion in clinical trials.N. Mattsson-Carlgren has served as a consultant for the Alzheimer's Disease Neuroimaging Initiative. A. Leuzy, S. Janelidze, S. Palmqvist, E. Stomrud, and O. Strandberg report no disclosures. Dr. Smith has served as a non-paid consultant for Roche. O. Hansson has acquired research support (for the institution) from Roche, GE Healthcare, Biogen, AVID Radiopharmaceuticals, and Euroimmun, and in the past 2 years, he has received consultancy/speaker fees (paid to the institution) from Biogen and Roche.


CYP2D6 gene influences endoxifen formation and thereby potentially therapy outcome. However, the association between CYP2D6 genotype and clinical outcome on tamoxifen is still under debate, as contradictory outcomes have been published. This review describes the latest insights in both CYP2D6 genotype and endoxifen concentrations, as well CYP2D6 genotype and clinical outcome, from 2018 to 2020.Tamoxifen is an important adjuvant endocrine therapy in estrogen receptor (ER)-positive breast cancer patients. It is mainly catalyzed by the enzyme CYP2D6 into the most active metabolite endoxifen. Genetic variation in the CYP2D6 gene leads to altered enzyme activity, which influences endoxifen formation and thereby potentially therapy outcome. The association between genetically compromised CYP2D6 activity and low endoxifen plasma concentrations is generally accepted, and it was shown that tamoxifen dose increments in compromised patients resulted in higher endoxifen concentrations. However, the correlation between CYP2D6 genotype and clinical outcome is still under debate. This has led to genotype-based tamoxifen dosing recommendations by the Clinical Pharmacogenetic Implementation Consortium (CPIC) in 2018, whereas in 2019, the European Society of Medical Oncology (ESMO) discouraged the use of CYP2D6 genotyping in clinical practice for tamoxifen therapy. This paper describes the latest developments on CYP2D6 genotyping in relation to endoxifen plasma concentrations and tamoxifen-related clinical outcome. Therefore, we focused on Pharmacogenetic publications from 2018 (CPIC publication) to 2021 in order to shed a light on the current status of this debate.Tamoxifen is a major option for adjuvant endocrine treatment in estrogen receptor (ER) positive breast cancer patients. The conversion of the prodrug tamoxifen into the most active metabolite endoxifen is mainly catalyzed by the enzyme cytochrome P450 2D6 (CYP2D6). Genetic variation in the  For many years, tamoxifen has been known as the most important adjuvant endocrine treatment in patients with estrogen receptor (ER) positive breast cancer ,2. It isCYP2D6 leads to altered enzyme activity and thereby potentially to an affected efficacy [HLA-B*5701 genotyping for abacavir, CYP2C19 genotyping for clopidogrel, and DPYD testing for fluoropyrimidines treatment [CYP2D6 genotyping for optimizing tamoxifen therapy based on pharmacokinetic studies [CYP2D6 genotype and outcome in breast cancer patients treated with adjuvant tamoxifen therapy [CYP2D6 status affects clinical outcomes in tamoxifen therapy [CYP2D6 genotyping for guiding adjuvant tamoxifen therapy, thus supporting the importance of the CYP2D6 genotype in tamoxifen therapy. However, the European Society for Medical Oncology (ESMO) indicated in a publication in 2019 that according to their view, there was no place for CYP2D6 genotyping in a clinical setting [As genetic variation in efficacy , pharmacreatment ,12,13. T studies  or outco studies ,16 were  studies ,18. The  therapy . This tr therapy ,22,23,24 therapy ,27,28,29CYP2D6 genotyping in breast cancer patients treated with tamoxifen. In addition, we included some other factors influencing plasma concentrations of tamoxifen and its metabolites. We searched for relevant studies published from 2018 (publication CPIC) to 2021 on CYP2D6 genotype in relation to endoxifen levels and tamoxifen-related clinical outcome.According to predictions, over 3 million women will be diagnosed with breast cancer in 2040 , which tN-demethylated, predominantly by CYP3A4 and CYP3A5, resulting in inactive N-desmethyltamoxifen (NDM-TAM)  and endoxifen show the strongest affinity with the ER ,31. As tNDM-TAM) . 4-hydrondoxifen ,31. As sndoxifen ,31. As Nndoxifen .CYP2D6 gene is located on chromosome 22q13.2 and is highly polymorphic [CYP2D6 gene deletions and CYP2D6 gene duplications, also occur [CYP2D6 is involved in the metabolism of ≈25% of the most commonly used drugs, whereas it only accounts for approximately 2% of total liver CYP protein capacity . As showymorphic . Currentymorphic . Geneticso occur ,36. Thisso occur ,36. Anotso occur . Geneticso occur ,37. The so occur .CYP2D6 genotype into predicted CYP2D6 phenotype is shown in CYP2D6*1/*4 interpretation from an Extensive/Normal metabolizer phenotype  into an Intermediate Metabolizer phenotype (definition used by the Dutch Pharmacogenetics Working Group (DPWG), which is mostly used in Europe). The second important change concerns the CYP2D6*10 allele, which was downgraded from AS = 0.5, comparable to other decreased activity alleles such as *9 and *41, to AS = 0.25 [A translation of S = 0.25 ,39.CYP2D6 genotype is not solely responsible for systemic endoxifen levels. In line with this recommendation, several authors [Most recently, Lee et al. suggested a dichotomization into normal and slow metabolizer CYP2D6 groups in an effort to improve and simplify the current system . Nonethe authors ,42,43,44 authors  suggest  authors ,46,47. T authors . TherefoCYP2D6 was associated with plasma concentrations of endoxifen.In addition to CYP3A4/5 and CYP2D6, other CYP enzymes such as CYP2C9, CYP2C19, and CYP3A4 are involved in the tamoxifen metabolism. It is shown that genetic variation in these genes affects tamoxifen and metabolite plasma concentrations ,56,57,58https://www.trialregister.nl/trial/8444, accessed on 28 November 2020). It is important to keep in mind that many enzymes may simultaneously influence the plasma levels of tamoxifen and its metabolites and therefore may also influence tamoxifen treatment efficacy.The most important phase II enzymes in tamoxifen metabolism are uridine 5′-diphospho-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), primarily SULT1A1 ,63. AssoCYP2D6 non-functional alleles, but also in carriers of two reduced function alleles, significantly reduced endoxifen levels were observed [*41/*41 or the combination *41/non-functional (defined as IM).Madlensky et al.  reportedobserved . A totalCYP2D6 genotype, with the strongest effect for endoxifen concentrations. PMs carrying two non-functional alleles had 4.5 to 5.5 times lower endoxifen concentrations than NMs carrying two functional alleles. This study was conducted in 192 Brazilian breast cancer patients with endoxifen concentrations measured at 3, 6, and 12 months after the start tamoxifen treatment. Additionally, CYP2D6 phenotypes significantly predicted tamoxifen metabolite levels across all time points in multivariate analyses [Nardin et al.  also conanalyses .Taken together, these studies ,71 confin = 15) and AS = 0–0.5 (n = 2) received dose-adjusted therapy of 30 and 40 mg/day, respectively. Before dose escalation, the mean endoxifen concentration differed significantly between genetic phenotype groups, and a correlation between plasma endoxifen concentrations and CYP2D6 activity was observed, although considerable inter-individual variability within the metabolizer groups was present [Breast cancer patients with lower endoxifen levels than the published efficacy threshold of 5.9 ng/mL might benefit from a higher dose tamoxifen than the standard 20 mg/day. Khalaj et al.  performe present . Dose ad present . This reCYP2D6 genotype-guided tamoxifen dosing in a randomized, open-label, phase II study, in which 186 Japanese breast cancer patients were genotyped. Patients carrying at least one CYP2D6 variant allele were randomly assigned to two groups: regular tamoxifen dosage  and increased tamoxifen dosage . This resulted in a significantly higher median serum through endoxifen concentration in the ID group as compared to the regular dose (RD) group [n = 11) and subsequently to 60 mg/day (n = 8), and NM patients were treated with the standard tamoxifen dose. After increase of the dosage, no significant increase in the occurrence of the most common side effect (hot flashes) or severe side effects was found [Tamura et al.  investigD) group . In 2011D) group  also demD) group  performeD) group ,75. In 2D) group  showed sas found ,73. Meanas found . This fias found ,75,76, wCYP2D6 polymorphisms, and endoxifen concentrations are determined 3 months after the start of tamoxifen treatment . Patients with endoxifen levels below 16 nM (≈5.97 ng/mL) receive a dose adjustment of tamoxifen. Subsequently, endoxifen concentrations were monitored during a period of 2 years. The first positive results have recently been presented at the ESMO virtual congress 2020 and confirm that TDM-guided dosing is feasible in order to optimize endoxifen levels [Currently, in the Netherlands, a large prospective clinical trial is ongoing in early stage ER-positive breast cancer patients, in order to assess the feasibility of TDM-guided dosing of tamoxifen in clinical practice (the TOTAM study). In this study, patients are genotyped for n levels .CYP2D6 genotype and adherence behavior explained 40% of endoxifen plasma concentration variability at 12 months [The plasma concentration of endoxifen is affected by many factors . An impo2 months .He et al.  investigDrug–drug interactions (DDIs) represent one of the major confounding factors for the evaluation of drug efficacy, and their clinical relevance is not always thoroughly investigated ,80. As tCYP2D6 genotype, and measured plasma concentrations of tamoxifen and its metabolites. It seems endoxifen plasma concentrations were significantly lower in CYP2D6 NMs with the concomitant use of (strong) CYP2D6 inhibitors as compared to those without the concomitant use of CYP2D6 inhibitors  [Borges et al.  prospectctively) . Similarctively) . In thisctively) . In a prctively) . Measurictively) . In addiThese studies indicate an important role of DDIs on tamoxifen activation. Treating physicians should take this into account in order to minimize deleterious interactions that may reduce drug efficacy .CYP2D6 genotype and additional factors on plasma concentration endoxifen described in this section are summarized in Studies regarding the CYP2D6 genotyping is improving tamoxifen treatment outcome. As summarized in 2009 by Dezentje et al. [CYP2D6 and tamoxifen was published [CYP2D6 genotyping prior to tamoxifen therapy as assessed by this international group of experts. However, in 2019, the European Society for Medical Oncology (ESMO) strongly argues against the use of the CYP2D6 genotype for determining tamoxifen dosage in a clinical setting [CYP2D6 and clinical outcome to see if new information has arisen.The main goal of preemptive e et al. , studiesublished , indicat setting . We reviCYP2D6 genotype was associated with breast cancer recurrence in patients treated with tamoxifen in an adjuvant setting. The CYP2D6 genotype was determined in two groups of predominantly non-Hispanic white patients; 1514 contralateral breast cancer (CBC) cases and 2203 unilateral breast cancer controls. CYP2D6 NM patients with a first breast cancer and treated with tamoxifen had nearly a 40% lower risk of CBC compared to patients without tamoxifen treatment . The risk of CBC in IMs and PMs (AS < 1) was not reduced by tamoxifen treatment  as compared to IMs and PMs without tamoxifen treatment [CYP2D6 copy number variations is an important limitation of this study; thereby, no identification of UMs was possible within the study population.In 2018, Brooks et al.  showed treatment . DespiteCYP2D6 polymorphisms on tamoxifen response after 6 months of tamoxifen treatment. Thirty carriers of CYP2D6 variant alleles had progression within 6 months after the start of tamoxifen treatment, whereas fourteen patients achieved good clinical tamoxifen response after 6 months [CYP2D6 variant alleles have worse 6-month prognosis after tamoxifen treatment. A limitation of this study is the limited amount of allele variants (n = 4) genotyped, thereby increasing the chances of missing patients carrying non wild-type alleles. Tamoxifen is used as a treatment option for metastatic breast cancer instead of adjuvant therapy.A prospective cohort study in 157 Egyptian metastatic breast cancer patients investigated the influence of 6 months . This suCYP2D6 genotype and breast cancer prognosis in 1309 Swedish breast cancer patients treated with tamoxifen. HRs were calculated in order to determine the association between CYP2D6 metabolizer status and breast cancer-specific mortality. This resulted in an HR of 2.59 for PMs (95% CI 1.01–6.67), 1.48 for IMs (95% CI 0.72–3.05), 1 for NMs (reference), and 4.52 for UMs (90% CI 1.42–14.37) [He et al.  examined2–14.37) . Thus, b2–14.37) ,71. Unli2–14.37) . Of note2–14.37) . There w2–14.37) . Due to CYP2D6 genotyping, patients were classified into five different groups; UMs, NMs, hetEM (heterozygous EMs), IMs, and PMs. Patients with two fully active alleles were classified as NM, whereas patients with one fully active and one non-functional allele were classified as hetEM. The 3-year relapse-free survival (RFS) was compared between the combined group of PMs, IMs, and hetEMs (n = 316), and the combined group of NMs and UMs (n = 322). Univariable analysis showed no difference in RFS between the two combined groups [CYP2D6 genotype on breast cancer-free survival (BCFS) was also reported by Rangel-Méndez et al. [n = 56) and PM/IM group (n = 15) [In 2019, Sanchez-Spitman et al.  conducted groups . Classifd groups . This std groups , Brauch d groups , and Brad groups  stated td groups . In addid groups . Moreoved groups . Gusellad groups  stated tz et al. . In this(n = 15) . The lim(n = 15) .CYP2D6 genotype and recurrence-free survival (RFS). Patients with AS = 0 (PMs) and patients with AS > 0  were compared; however, no association of CYP2D6 status with RFS was found. After adjustment for relevant clinical covariates, analyses showed that a higher AS was associated with inferior RFS . In 476 breast cancer patients who did not receive adjuvant tamoxifen therapy, an AS > 0, as well as an increasing AS, were associated with superior RFS .Hertz et al.  conducteCYP2D6 genotype-guided tamoxifen dosing on progression-free survival (PFS) in 186 Japanese breast cancer patients. The PFS rates at 6 months did not differ between the increased dosage (ID) group (67.6%) and the regular dose (RD) group (66.7%). PFS curves of both arms did not significantly differ [CYP2D6 variant alleles. This resulted in combining PMs and IMs in the intervention arm.Tamura et al.  examinedy differ . The 6 my differ  and Rangy differ , they diCYP2D6 and clinical outcome described in this section are summarized in Publications from 2018 to 2021 on CYP2D6 genotype, additional factors also seem to influence endoxifen levels, such as treatment adherence and drug–drug interactions, for example caused by the concomitant use of (strong) CYP2D6 inhibitors. In the last years, various prospective and retrospective studies were performed to examine the association between CYP2D6 genotype and prognosis in (ER-positive) breast cancer patients, which led to, again, contradictory results. Some of these studies have been criticized, mainly because they were neither designed nor statistically powered to answer specific pharmacogenetic questions. Between 2018 and 2021, two studies provided evidence for a relation between CYP2D6 genotype and breast cancer recurrence as well as breast cancer-specific mortality after tamoxifen treatment. In that same period, four studies reported no association with clinical outcome in tamoxifen treatment. Therefore, it seems that the controversy regarding the association of CYP2D6 genotype and tamoxifen-related clinical outcome continues. In our opinion, this controversy will exist up until large randomized control trials will be performed.CYP2D6 is the main enzyme in the conversion of the prodrug tamoxifen into its most important metabolite endoxifen. Currently, a plasma concentration endoxifen of 5.97 ng/mL is maintained to indicate the sufficient efficacy of tamoxifen treatment. Patients with reduced CYP2D6 activity due to genetic variations often fail to reach this limit. Tamoxifen dose increments in these CYP2D6 compromised patients result in higher plasma concentrations of endoxifen without a higher frequency of ADRs. In addition to the


MTHFR) C677Tand methionine synthase reductase (MTRR) A66G genotypes are associated with male infertility in Iranian men andto evaluate its effect on seminal levels of folate and vitamin B12.The relation between key enzymes in regulation of folate metabolism and male infertility is the subjectof numerous studies. We aimed to determine whether 5, 10-methylenetetrahydrofolate reductase  and 77 normozoospermic men who attended Avicenna infertility clin-ic. Single nucleotide polymorphism (SNP) analysis was carried out in genomic DNA by polymerase chain reaction(PCR)-restriction fragment length polymorphism (RFLP) method for MTHFR C677T, our founding showed that T carrier was conversely lower in OAT than normozoospermic men whereas in MTRR A66G, A and G carrier showed no significant difference between the two groups. The concentration of seminal folate was not different between normozoospermic (18.83 ± 17.1 ng/ml) and OAT (16.96 ± 14.2 ng/ml) men (P=0.47). The concentration of vitamin B12 was slightly higher in normozospermicmen (522.6 ± 388.1 pg/ml) compared to OAT men .In 12. The present study showed that two SNPs of MTRR A66G and MTHFR C677T cannot be seen as a risk factor formale factor subfertility.The MTHFR C677T and MTRR A66G have no effect on the concentrations of seminal folate and vitaminB MTHFR gene is located onchromosome 1 (1p36.22) (MTRR) gene, onchromosome 5, also plays a vital role in DNA synthesis. It is well known that folate andmethionine metabolism play essential roles in both DNA methylation and synthesis .ynthesis .MTHFR and MTRR play key interrelated roles in folatemetabolism. MTHFR catalyzes the regulation of cellularmethylation through the conversion of 5, 10-methylenetetrahydrofolate (THF) to 5-methyl-THF, the primary circulating form of folate metabolism. MTRR is required forthe reductive methylation of vitamin B12, also known ascobalamin, an activated cofactor for methionine synthase(MTR), which catalyzes the methylation of homocysteineto methionine  (The homozygous C667T in theascular) , cancersascular) . Previouascular) .MTRR gene is A66G, whichresults in an amino acid substitution from methionine to isoleucine at codon 22 (M22I)  levels  .MTRR 66) levels .MTHFR gene, 677C>T, 1298A>C and the MTRR gene, 66A>G, areassociated with male infertilityFunctional studies indicated that individuals possessing both mutations showed the lowestenzyme activities . As a re MTHFR gene 677C>T, as well asMTRR gene 66A>G in men with OAT and normozospermia. Furthermore, weinvestigated the correlation of these genetic variants with seminal folate and vitamin B12levels. In the previous study, we demonstrated that administration of folic acid andco-administration of folic acid and zinc sulphate during randomized, double-blind dietaryprogram did not improve the quality of sperm in infertile men with severely compromisedsperm parameters, oligoasthenoteratozoospermic (OAT) . The pre6 ml-1, sperm motility <50% and normal sperm morphology <30% were included as OAT and infertile men with normalsperm parameters were classified as normozospermia.In this retrospective study, semen analysis was done for 254 men with OAT and 77 men withnormozospermia who attended Avicenna Infertility Clinic . In allsamples, semen parameters, sperm concentration, sperm motility, viability and morphologywere evaluated in accordance with the WHO guidelines  and they6/ml), varicocele, chronic systemic diseases, autoimmune disorders or history of smoking,in addition to excessive alcohol and drug consumption were excluded from the study.Patients with leukocytospermia was allocated for our project. All procedures were done inaccordance with the ethical Helsinki standards. The semen samples were collected by masturbationonly 48-72 hours after sexual abstinence and ejaculatedinto a clean plastic specimen cup. Samples were deliveredto the laboratory within 1 hour of collection at 20-40°C.Following complete liquefaction, standard semen analysis was conducted for all participant according to WHOguidelines . The remGenomic DNA was extracted from white blood cellsaccording to sodium salting out extraction method. TheDNA purity and concentrations were determined bymeasurement of absorbance at 260/280 nm. MTHFR gene was studied by polymerase chainreaction (PCR)-restriction fragment length polymorphism (RFLP) method. The C677T SNP (rs#1801133) ofWe employed theF: 5'-CATCCCTATTGGCAGGTTACCC-3' and R: 5'-TGCGAGGACGGTGCGGTGAGA-3'PCR primers which were designed using Gene Runnersoftware. PCR cycles were as follow: 32 cycles of 94°Cfor 30 seconds, 58.4°C for 30 seconds and 72°C for 30seconds. A 271 bp PCR product was digested overnight byHinfI restriction enzyme endonuclease  with the recognition sequence 5'-GˆAGTC-3'at 37°C. The PCR products were visualized on 2% agarosegel. A 271 bp fragment was seen in the absence of mutation [wild-type (CC)], 271, 177 and 94 bp fragments wereobserved in the presence of one allele mutation [heterozygotes (CT)], and 177 and 94 bp fragments were seen in thepresence of two allele mutations [homozygotes (TT)].MTRR A66G polymorphisms. The PCR-RFLP assay was employed for identifying MTRR A66G: The primers for F: 5'-AGGCAAAGGCCATCGCAGAAGACAT-3' andR: 5'-GGCTCTAACCTTATCGGATTCACTA-3' were designed using Gene Runner software. The forwardprimer was comprised of a mismatch (underlined base C inthe primer sequence), generating an NdeI restriction site,5'-CAˆTATG-3', when the polymorphic allele was present.PCR cycles were as follow: 35 cycles of 94°C for 30 seconds, 58.4°C for 30 seconds and 72°C for 30 seconds. Theanticipated PCR product of 98 bp was digested into fragments of 74 and 24 bp by NdeI restriction enzyme endonuclease  in the presence of the G allele but remains uncut in the presence of the A allele . It mean12 levels were measured using radioimmunoassay (RIA)method according to the manufacturer’s instructions .Semen folate and vitamin B MTHFR C>T and MTRR A>G mutations innormozoospermic and OAT groups. Folate and vitamin B12 concentrations in two studiedgroups were compared using parametric tests such as Independent-Samples t test andunconditional logistic regression model to calculatethe odds ratios (OR) and 95%confidence intervals (95% CI). Fisher’s exact test was employed in analyzing associationof MTHFR and MTRR mutations with semen concentrationof folate and vitamin B12. Differences were considered significant if P valuewas<0.05. Results of the two groups were compared using SPSS 16.0 for Windows software . TwoSample Kolmogorov-Smirnov test (K-S test) revealed that our datafollow a normal distribution and thus parametrical test was employed. Monte Carlo test wasconducted to compareDemographic characteristic information, including ageand infertility period in OAT and normozoospermia men,are given in Figure 1 shows the results of PCR-RFLP for MTHFRC677T and MTRR A66G gene polymorphisms withregard to digestion using HinfI and NdeI, respectively.  MTHFR gene polymorphism indicated that frequency of CCgenotype in OAT men was higher than in men with normozoospermia (59.8 vs. 42.9%), while thefrequency of CT genotype was lower in OAT men (46.8 vs. 34.3%). In addition, frequency of TTgenotype was lower in OAT men . Frequency of AG genotype in the two studiedgroups was almost identical (61 vs. 60.6%) and frequency of GG genotype in OAT group washigher than normozoospermic group (17.4 vs. 13%).As given in Table 2, analysis of MTHFR C677T between normozoospermic and OAT groups showed that T carrier wasconversely lower in OAT group than normozoospermic group .Additionally, our analysis did not show significant difference  in A(AA) and G carrier (AG+GG) from MTRR A66G between normozoospermic men andOAT men  and T carrier (CT+TT) in12 concentrations were identified in 19.5 and 20.5% of thenormozoospermic and OAT men, respectivelyAccording to Furthermore, seminal folate and vitamin B12 concentrations were compared between C and Tcarrier in MTHFR C677T. They were also compared between A and G carrier in MTRR A66G, whilenone of them was statistically different . MTHFR suffered from severe reproductive defects. Inthese mice, spermatogenesis was failed during early postnatal development and resulted intotal infertility  concentrations and any semen parameters in fertile andinfertile men. They also found that infertile men had lower serum folate concentrations thanfertile men, but there was no significant difference in red blood cell folate (RCF),B12 or tHcy. However, another study showed that adequate intake of vitamins B9and B12 affects sperm parameters in men with different MTHFRpolymorphisms, especially T allele genotypes (The current study also demonstrated no association betweeny etal.  while noenotypes .As anticipated, OAT men had overall poorer semenparameters than normozoospermic controls. Theresults showed that infertile subjects had lower semenconcentration, greater percentage of abnormal spermmorphology, higher percentages of non-motile spermand more incidences of DNA fragmentation than fertilecontrols.Some limitations of this study were low number ofnormozoospermic men in comparison with OAT men andabsence of some demographic data regarding individualsthat participated in the current study. MTHFR C677T and MTRR A66G genepolymorphisms as risk factor for male factor subfertility in Iranian population. Inaddition, there is no statistical difference in seminal vitamin B12 and folateconcentrations between normozoospermic and OAT men. Moreover, this study indicated noassociation between MTHFR C677T and MTRR A66Gpolymorphisms with seminal vitamin B12 and folate concentrations innormozoospermic and OAT men. However, larger sample size and well-designed studies areneeded to compare the effect of other folate-related enzyme genes in the Iranian population.Larger sample size could generate better statistical results and sufficient data to confirmthe improvement of secondary outcomes.The present study donot reveal the


Increased vascular permeability is a hallmark of several cardiovascular anomalies, including ischaemia/reperfusion injury and inflammation. During both ischaemia/reperfusion and inflammation, massive amounts of various nucleotides, particularly adenosine 5′-triphosphate (ATP) and adenosine, are released that can induce a plethora of signalling pathways via activation of several purinergic receptors and may affect endothelial barrier properties. The nature of the effects on endothelial barrier function may depend on the prevalence and type of purinergic receptors activated in a particular tissue. In this review, we discuss the influence of the activation of various purinergic receptors and downstream signalling pathways on vascular permeability during pathological conditions. The vascular endothelium (VE), consisting of monolayers of endothelial cells (ECs), is located at the interface between the vascular and perivascular compartments and extends over a wide surface area. The VE separates strictly two compartments and regulates the trafficking of ions, solutes, macromolecules and leukocytes across the vessel wall, thus maintaining tissue homeostasis ,2. AdditEndothelial barrier integrity is maintained by the equilibrium of competing adhesive and contractile forces generated by adhesive molecules located at cell–cell and cell–matrix contacts and the acto-myosin-based contractile machinery, respectively . ECs areTwo members of the Rho family of GTPases, RhoA and Rac1, are the major regulators of endothelial actin cytoskeleton dynamics and contraction and thereby play a key role in the maintenance of endothelial barrier integrity. Constitutive activation of RhoA results in the loss of basal VE-cadherin and potentiates hypoxia-reoxygenation (H/R)-induced loss of endothelial barrier function, whereas suppression of RhoA activity attenuates the agonist-induced increase in endothelial permeability ,9. On th2+ from intracellular stores [The activation state of the endothelial contractile machinery is regulated by the phosphorylation state of regulatory myosin light chains (MLC), which are phosphorylated by MLC kinase (MLCK)  and dephr stores ,14,15. BEndothelial barrier integrity is influenced by several circulating, blood-borne hormones and agents/factors such as adenosine triphosphate (ATP) and its metabolites adenosine diphosphate (ADP) and adenosine. The major sources of vascular nucleotides are erythrocytes, platelets, and the endothelium . Platele1, A2, and A3 subtypes, depending on their affinity for adenosine. P2 receptors are further classified into ionotropic P2X and metabotropic P2Y receptors [There are two main classes of purine receptors: P1 receptors activated by adenosine and analogues, and P2 receptors recognised by purine and pyrimidine nucleotides , uridine diphosphate (UDP)). P1 receptors are further divided into Aeceptors ,28. Nineeceptors . Nearly eceptors ,29. Seveeceptors ,31. Thiseceptors .1, A2A, A2B, and A3, which are classified as high  or low (A2B) affinity for binding their parent physiological agonist, adenosine [1 and A3 receptors are coupled to Gq and/or Gi/o, whereas A2A and A2B are coupled to Gs proteins. Activation of A1 and A3 receptors results in inhibition of adenylyl cyclase (AC) activity, leading to reduction in cyclic AMP (cAMP) production and suppression of downstream signalling [3-dependent release of Ca2+ from the endoplasmic reticulum (ER), protein kinase C (PKC) activation, and nitric oxide (NO) production [1 adenosine receptors stimulates the opening and blockade of K+ channels and P- and N-type Ca2+ channels, respectively [1 and A3 receptors leads to PKC-dependent and independent mitogen-activated protein kinase (MAPK) activation [2A and A2B receptors results in activation of AC, enhanced cAMP production, and activation of downstream signalling [Physiological extracellular adenosine levels range from 20 to 300 nM, which rise to a low micromolar range during exercise and to a high micromolar level under pathological conditions like ischaemia ,34. Undedenosine . All foudenosine . The A1 gnalling ,38. Theioduction ,40,41,42ectively ,44. Actitivation . Activatgnalling . Adenosignalling ,46.Adenosine is a non-selective agonist for all adenosine receptors and produces differential effects on endothelial permeability of various vascular beds depending on the type of receptors expressed.2A or A2B receptors seem to mediate the protective effects of adenosine in the lung [2-receptor agonist reduced the IR-induced increase in microvascular permeability [2A and A2B receptors protected against hypoxia and lipopolysaccharide (LPS)-induced development of lung oedema [2A or A2B receptors in mice resulted in loss of adenosine-mediated preservation of the lung microvascular endothelial barrier [2B receptors in these mice did not rescue but worsened the conditions, which were accompanied by enhanced loss of pulmonary barrier function [2B receptors. In contrast to murine lungs, in feline lungs, adenosine A1 receptor activation mediates IR- and LPS-induced pulmonary microvascular barrier disruption [1 receptor antagonists xanthine amine congener (XAC)/8-cyclopentyl-1,3-dipropylxanthine (DPCPX) ameliorates IR-induced lung injury and oedema in these animals. These species differences are probably due to differential expression of adenosine receptors in murine and feline lungs. Like A2 receptor activation, pharmacological activation of adenosine A3 receptors with a selective agonist also protects against reperfusion-induced lung oedema. This protective effect is lost in A3 knockout mice in vivo [In the lung vasculature, adenosine signalling has largely been shown to enhance endothelial barrier properties and ameliorate agonist-induced hyperpermeability. In a mouse model of acute lung injury, knockdown of CD39 or inhibition of CD73, the two sequential enzymes responsible for adenosine production, resulted in development of severe lung oedema in response to ventilation compared with wild-type littermates. These animals were rescued by the addition of exogenous apyrase, suggesting a protective role played by adenosine . Both adthe lung ,49. In aeability . Pharmacg oedema ,52, wher barrier ,52. Thes barrier . Likewis barrier . On the  barrier . Howeverfunction , suggestsruption ,58: perf in vivo . However1 and A2A receptors [N-ethylcarboxamidoadenosine (NECA) and selective A1 and A2A receptor agonists increased blood–brain barrier permeability to low-molecular-weight dextran [1 or A2A receptors [2A receptors protected mice against thromboembolic stroke-induced development of cerebral oedema and leukocyte infiltration [2A receptor agonist used clinically as a coronary vasodilator for myocardial perfusion imaging, increased permeability of the human blood–brain barrier in vitro [The blood–brain barrier is a highly specialised structure formed by a very tight monolayer of microvascular ECs that are distinct from ECs of other vascular beds . The braeceptors ,62,63. Aeceptors . Accordiltration . Likewisin vitro  and in tin vitro ,68. Sevein vitro ,72,73,742 receptor activation increased permeability of rat coronary microvascular ECs in vitro [2 receptors were also antagonised. In line with these reports, we have previously demonstrated that reperfusion caused the release of ATP from isolated rat coronary microvascular ECs that was degraded to adenosine. Inhibition of either ectonucleotidases or adenosine receptors abrogated endothelial barrier failure, whereas addition of apyrase and ectonucleotidases worsened reperfusion-induced endothelial barrier failure [2 receptors) in coronary microvascular ECs [As in the blood–brain barrier, adenosine receptor activation in the coronary microvasculature results in loss of barrier integrity. Ain vitro . Infusioin vitro . Similarin vitro . However failure . In a fo failure . In a reular ECs  that cauular ECs . This isular ECs ,82. Inhiular ECs ,80. Acti2A and A2B receptors by adenosine and its analogues. Enhanced cellular cAMP levels suppress the activity of the endothelial contractile machinery in a RhoA/Rock-dependent manner and activate Rac1 GTPase via protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) activation [In general, adenosine receptor activation in macrovascular ECs enhances endothelial barrier properties and ameliorates the effect of barrier-disrupting agents ,83,84,85tivation . Table 1+, K+, and Ca2+ under physiological conditions, although a recombinant P2X5 receptor has been shown to allow the passage of Cl¯. Excitable cells are thus depolarised upon activation of P2X receptors. Moreover, increased intracellular Ca2+ levels initiate a diverse array of Ca2+-dependent signalling pathways, both in excitable and non-excitable cells, that regulate various cellular processes, including cell migration, proliferation, necrosis, and apoptosis.The family of P2X receptors are non-selective ion channels comprising one or more of seven monomeric proteins (P2X1–P2X7). Each monomeric P2X protein consists of two transmembrane domains (TM1 and TM2) linked via an extracellular ligand-binding loop. The monomeric P2X proteins combine to form trimeric homomultimeric or heteromultimeric ion pores ,97,98,992+ currents in endothelium [(−/−) mice do not show an EC response to flow, such as calcium influx and subsequent production of NO [P2X receptors are widely expressed throughout the cardiovascular system. mRNA and protein of all P2X receptors have been detected in the endothelium of various types of blood vessels ,106,107,othelium  that mayothelium . The veson of NO . A loss-on of NO . Cardiacon of NO . Accordion of NO . In HUVEon of NO . In thison of NO . A high-on of NO . In lineon of NO . Differe(−/−) mice were protected against traumatic brain injury-induced development of brain oedema [Unlike P2X4 receptors, activation of P2X7 receptors in ECs is primarily linked to a proinflammatory and hyperpermeability response. In an in vitro model of the blood–brain barrier, ATP induced an increased production of matrix metallopeptidase 9 (MMP9) in an interleukin (IL)-1β-dependent manner, which was responsible for the degradation of tight junction proteins . These An oedema  and alson oedema . In contn oedema  reportedn oedema .1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14) have been cloned and are further classified into two sub-families based on sequence similarities and signal transduction pathways [1-like subfamily includes the P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors that are coupled to Gq proteins. The P2Y11 receptors are coupled additionally to Gs proteins, activation of which leads to an activation of AC and enhanced production of cAMP [12-like subfamily includes P2Y12, P2Y13, and P2Y14 receptors, which mediate cellular signalling via Gi proteins [P2Y receptors are membrane-bound class A GPCRs for extracellular nucleotides . At prespathways ,127,128. of cAMP . The P2Yproteins ,130.1 receptor is a ubiquitously expressed endothelial purinergic receptor on most EC types. It is a Gq-linked GPCR that has been well-studied in platelet biology, for which ADP acts as a natural agonist and ATP an antagonist [1 knockout mice are viable, fertile, normal in size, and do not present gross physical or behavioural abnormalities [1(−/−) homozygous mice are more susceptible to lung infections and are resistant to ADP/collagen-induced thrombin formation [1(−/−) apoE(−/−) double knockout mice have reduced amounts of atherosclerotic lesions [1 receptor may be involved in the atherogenic response [1 knockout as well as P2Y1 receptor antagonist-treated ECs in vitro [1 receptor may potentiate vascular inflammation and hyperpermeability. However, in a mouse model of traumatic brain injury, development of cerebral oedema was ameliorated in mice treated with the P2Y1 agonist 2-methylthioadenosine 5′diphosphate (2MeSADP). These protective effects of the P2Y1 agonist were lost in inositol 3-phosphate receptor 2 (IP3R2)-knockout mice, suggesting that it is an IP3/Ca2+-dependent phenomenon [1 mRNA is expressed in HUVECs, and treatment of cultured HUVEC monolayers with ADP as well as P2Y1-selective agonist 2MeSADP antagonised thrombin-induced HUVEC hyperpermeability  mice results in reduced inflammatory response and increased plaque stability [2 receptor activation under inflammatory conditions. Accordingly, knockdown of P2Y2 receptors in HUVECs ameliorated LPS-induced transendothelial migration of activated neutrophils [P2Y and ATP . Global rtension . However vessels , and endtability , suggesttrophils .4-null mice are viable but display microcardia , suggesting that the P2Y4 receptor plays a role in postnatal heart development [4 receptor, and loss of the P2Y4 receptor in cardiac ECs results in reduced growth and migratory capacity in vitro [4 knockout mice are protected from myocardial ischaemic injury, cardiac inflammation, and fibrosis in a left anterior descending (LAD) coronary artery ligation model [4-knockout mice are protected from an LPS-induced cardiac microvascular hyperpermeability response. These data suggest that although the endothelial P2Y4 receptor is required for normal development of the heart in mice, its activation may induce vascular hyperpermeability under pathological conditions.P2Yelopment . Interesin vitro . Surprison model . Moreove6 is a Gq-coupled receptor activated by UDP [6 receptors results in macrocardia (larger heart), and mice lacking the P2Y6 receptor show an amplified pathological cardiac hypertrophic response [6 receptors results in reduced vascular inflammation and ameliorated neointima formation in an atherosclerosis mouse model [6 receptors with a selective antagonist aggravates development of cerebral oedema in a mouse model of ischaemic brain injury [P2Yd by UDP  that is d by UDP ,142. Gloresponse . Howeverse model ,144. In n injury .11 is the only known human P2Y receptor coupled to Gs [11 receptor does not exist or at least has not yet been identified. Moreover, the lack of selective agonists and antagonists for this receptor as well as specific detection tools (antibodies) make functional investigations of the P2Y11 receptor difficult [11 mRNA in HUVECs, but other EC types were not investigated. Presumably, if it is expressed in some EC type, one would expect its activation would raise intracellular cAMP levels that can interact with multiple signalling pathways, e.g., Rac1-dependent actin cytoskeleton rearrangement and MLCP-mediated inactivation of the contractile machinery, thus modulating endothelial barrier properties.P2Yed to Gs ,128,129.ifficult . We did 12-like subfamily comprises three members: P2Y12, P2Y13, and P2Y14. All of these receptors are coupled to Gi, and their activation leads to suppression of AC activity and cAMP production [12 is well-studied in relation to platelet biology, and its antagonists are used clinically as anticoagulants in various pathological conditions. In human cardiac-derived mesenchymal cells, ticagrelor, a P2Y12 receptor antagonist, induces the release of anti-apoptotic exosomes [12 [13 [12 receptor mRNA and protein in HUVECs, and a specific P2Y12 antagonist increased intracellular cAMP levels and protected against thrombin-induced hyperpermeability [13 but not P2Y14 mRNA in primary HUVECs (unpublished). In vasa vasorum ECs, ADP mediates a mitogenic response partly via P2Y13 receptors [14 receptor has been reported in rat primary brain microvascular ECs [14 receptor) mediated a contractile response in isolated pancreatic arteries in an endothelium-dependent manner, and this effect was abrogated by a selective P2Y14 receptor antagonist [13 and P2Y14 receptors in the control of endothelial barrier properties. The P2Yoduction . P2Y12 iexosomes  that mayomes [12 ,148 and  [12 [13  has beeneability . We alsoeceptors . The expular ECs , and acttagonist . No furt2A and A2B receptors, raises intracellular levels of cAMP in the lung microvascular bed and thus strengthens the barrier properties and ameliorates hypoxia- and inflammation-induced development of oedema. Selective agonists for adenosine A2 receptors are available that may be tested  for clinical use in various oedematous abnormalities of the lung, e.g., acute lung injury. Conversely, A2 receptor activation in brain and coronary microvasculature results in transient opening of the cell–cell junction in a cAMP-dependent manner. This property of the brain microvasculature can be exploited for local delivery of drugs to the CNS. P2 receptors are also widely distributed in the vascular bed. Chronic P2X receptor activation leads to endothelial barrier destabilisation and oedema formation, an effect primarily attributed to the P2X7 receptors. There is a need for the development of more selective and potent P2X7 receptor antagonists to ameliorate inflammation-induced loss of endothelial barrier function. There is also a lack of selective P2Y receptor agonists and antagonists, which makes the investigation of P2Y receptors in relation to endothelial barrier function difficult. We and others have documented that ATP at low micromolar concentrations stabilises endothelial barrier function, mainly via activation of various P2Y receptors, whereas at high concentration (in the millimolar range), it may act as a danger-associated molecular pattern (DAMP) [12 receptor blocks inflammation-induced increases in endothelial permeability [Endothelial barrier properties are influenced by extracellular nucleotides via activation of various purinergic receptors. The response depends on the type of receptor(s) present and the local concentration of the nucleotides. Adenosine, primarily via activation of An (DAMP) , amplifyeability . Further


To investigate the role of m6A methylation in the expression of non-coding RNA and miRNA, we used a system of adenine base editors (ABEs). Here, we mutated regions up- and downstream of miRNA 675 m6A modification sites in the H19 locus using HEK293T, L02, MHCC97L, MHCC97H, A549, and SGC-7901 cells. Our results showed that a T–A base transversion had occurred in all cell lines. Moreover, mutation of the regions upstream of the miRNA 675 m6A modification site led to reduced expression of H19 and the induction of cell apoptosis in HEK293T cells. To further confirm our results, L02 and MHCC97L cells were detected using ABEs system. The results indicated increased cell apoptosis and reduced expression of miR675 as well as H19. To confirm the relationship between H19 and miR675 expression, overexpression and knockdown studies were performed. The results showed that reduced HI9 expression induced cell apoptosis through miR675. Taken together, these results indicate that m6A modification can regulate the expression of H19 and miR675 which induce cell apoptosis.Methylation of the adenine base at the nitrogen 6 position (m H19 plays a crucial role in the development of cancer [H19, has been shown to have an oncogenic role in liver cancers [H19 could induce cell apoptosis in A549, a lung cancer cell line [H19/miR675 axis can regulate cell apoptosis [H19 or miR675 can influence tumor cell behavior.The long noncoding RNA (lncRNA)  cancers ,3. Our p6A (methylation of the adenine base at the nitrogen 6 position) methylation plays an important role in the post-transcriptional modification of RNA [6A methyltransferases METTL3, METTL14, and WTAP methylate the N6 position of adenosine [6A demethylases FTO and ALKBH5 reverse the RNA methylation process [YTHDF2, as an m6A ‘reader,’ recognizes m6A sites on target mRNAs and regulates the mRNAs’ fate [6A modification in microRNA and lncNA affects cell development and fate [6A modification might have a role in noncoding RNA as well as miRNA.Recent reports have suggested that mn of RNA ,8. As ‘wdenosine ,10. As ‘ process ,12. FinaAs’ fate . Indeed,and fate . These d6A modification of miRNA 675 in the H19 locus. Moreover, cell apoptosis and m6A expression levels were evaluated in HEK293T, L02, and MHCC97L cells. The role of m6A modification in the expression patterns of miRNA and lncRNA was analyzed using the ABEs system.Currently, the CRISPR/Cas9 system is the most widely used gene-editing tool. It uses an RNA-guide Cas9 protein combined with a short RNA (sgRNA) to induce double-strand breaks in target genomic DNA . The ade2.HEK293T, L02, MHCC97L, MHCC97H, SGC-7901, and A549 cells were cultured in Dulbecco’s modified Eagle’s medium, high glucose , supplemented with 10% fetal bovine serum . The cells were maintained at 37°C in 5% CO6A modification of miR675 in the H19 locus (upstream of position: chr11:2018320 and downstream of position: chr11:2017630) was analyzed using the online software tool m6AVar (http://m6avar.renlab.org).ABE7.10 plasmids were obtained from Addgene (102919). The min vitro transcription have been described previously [Protocols for sgRNA design and the procedures required for eviously . The sgRd-lysine-coated plates (Corning) in the absence of any antibiotic. Twelve to fifteen hours after plating, cells were transfected with 750 ng of base-editor plasmid and 250 ng of guide RNA plasmid in the presence of 1 µl of Lipofectamine 2000 (Thermo Fisher Scientific).For cell transfection, HEK293T, L02, MHCC97L, MHCC97H, SGC-7901, and A549 cells were seeded into 48-well poly-H19 were obtained from RiboBio . The siRNA target sequence was GCGGGTCTGTTTCTTTACT. pcDNA3.1-H19 was purchased from GenePharma . miR675-3p-mimics and miR675-3p-inhibitor were obtained from RiboBio . HEK293T cells were transfected with si-H19, pcDNA3.1-H19, miR675-3p-mimics, and miR675-3p-inhibitor for 48 h, respectively. Control cells were transfected with nonspecific, scrambled siRNA.Synthetic RNA oligonucleotides targeting H19, miR675, and m6A-related gene expression using the BioEasy SYBR Green I Real-Time PCR Kit on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System . The miR675 3p and 5p sequences are listed in Supplementary Table S2, and the miRNA primer sequences are listed in Supplementary Table S3. The primer sequences of m6A-related genes and H19 are listed in Supplementary Table S4. For PCR, the initial denaturation was conducted at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s. The 2CT−ΔΔ method was used to determine relative gene expression. The experiments were performed at least in triplicates.Total RNA was extracted from cells using the AllPrep DNA/RNA Micro Kit  according to the manufacturer’s instructions. cDNA was synthesized using the First-Strand cDNA Synthesis kit . Quantitative real-time PCR (qRT-PCR) was performed to determine 6 cells/ml. For each treated cell sample, Annexin V-FITC and PI were added according to the manufacturer’s instructions. These cells were incubated for 30 min and then analyzed with an Accuri™ C6 flow cytometer .The procedure for cell apoptosis detection has been previously described . Briefly6A  antibodies and incubated at 4°C overnight. Following this, the cells were washed three times with PBS for 10 min each followed by incubation with Alexa Fluor 488–conjugated secondary (anti-rabbit) antibodies for 1 h at room temperature. DNA was stained with 10 ng/ml Hoechst 33342 (Thermo Scientific) for 15–20 min. The cells were then washed thrice with PBS for 10 min each, air-dried, and mounted on a coverslip and a glass slide using an antifade mounting medium . A confocal laser scanning microscope was used for imaging.Briefly, the cells were washed three times in PBS and then fixed with 4% paraformaldehyde for 30 min at room temperature. After fixation, the cells were washed again with PBS containing 0.2% Triton X-100 for 30 min. The cells were then incubated in PBS containing 1% bovine serum albumin (BSA) for 1 h. Next, the cells were probed with mt test (Unpaired t test) was used to analyze the data. A P-value <0.05 was considered statistically significant.All data were analyzed using GraphPad Prism 5.0 . A 6A modification in the expression of H19, the ABE7.10 system was used. The m6A modification site 129 bp upstream of miR675 in the H19 locus was mutated in HEK293T cells . The results showed that m6A expression was not altered in either L02 and MHCC97L cells  or A-to-G (ABE7.10) conversions ,20. In t6A modification in apoptosis, the expression patterns of H19 and miR675 were analyzed. A previous study suggested that m6A modification was important for the expression of lncRNA and miRNA [6A modification site of miR675 in the H19 locus were evaluated. The results demonstrated that mutations in the regions upstream of the m6A modification site could suppress the expression of H19 and miR675, whereas mutations in the regions downstream of the m6A modification site have no effect on the expression of H19 and miR675. To confirm the expression patterns of H19 and miR675 in apoptotic cells, overexpression and knockdown of H19 and miR675 were examined. Previous reports showed that the expression of H19 and miR675 was associated with cell apoptosis in cancer cells [H19 expression induced cell apoptosis through miR675, which was confirmed in human colorectal cancer cells [6A modification site play a role in regulating the expression of H19 and miR675 which can induce cell apoptosis.To further investigate the role of mnd miRNA . In our er cells ,23. Our er cells . These rH19 and miR675 expression led to increased p53 protein expression, which regulates cell apoptosis [6A modification site inhibited miR675 and H19 expression, inducing cell apoptosis, possibly through the p53 protein. Moreover, an abnormal expression pattern of m6A-related genes was observed in liver cancer cells, which was in accordance with previous data [ALKBH5 overexpression promotes invasion and metastasis in gastric cancer cells [ALKBH5 expression may regulate the demethylated process and play a role in HCC metastasis. In addition, the global m6A expression was maintained after mutations of the regions upstream of the m6A modification site in L02 and MHCC97L cells. These results suggested that miR675 was regulated by m6A modification and has a role in H19 expression, which in turn influences the fate of the cell.There is evidence that reduced poptosis . Our datous data . A previer cells . Indeed,6A site upstream of miR675 in the H19 locus. The expression of H19 and miR675 was reduced by targeted point mutations. These mutations also induced cell apoptosis. Overall, our data suggest that m6A modification plays a role in gene expression and cell apoptosis.In summary, the ABE7.10 system resulted in efficient T–A base conversion of the mClick here for additional data file.