Development and application of a population pharmacokinetic model repository for caffeine dose tailoring in preterm infants.

BACKGROUND: Considerable interindividual variability for the pharmacokinetics of caffeine in preterm infants has been demonstrated, emphasizing the importance of personalized dosing. This study aimed to develop and apply a repository of currently published population pharmacokinetic (PopPK) models of caffeine in preterm infants to facilitate model-informed precision dosing (MIPD). RESEARCH DESIGN AND METHODS: Literature search was conducted using PubMed, Embase, Scopus, and Web of Science databases. Relevant publications were screened, and their quality was assessed. PopPK models were reestablished to develop the model repository. Covariate effects were evaluated and the concentration-time profiles were simulated. An online simulation and calculation tool was developed as an instance. RESULTS: Twelve PopPK models were finally included in the repository. Preterm infants' age and body size, especially the postnatal age and current weight, were identified as the most clinically critical covariates. Simulated blood concentration-time profiles across these models were comparable. Caffeine citrate-dose regimen should be adjusted according to the age and body size of preterm infants. The developed online tool can be used to facilitate clinical decision-making. CONCLUSIONS: The first developed repository of PopPK models for caffeine in preterm infants has a wide range of potential applications in the MIPD of caffeine.

Population pharmacokinetics of valproic acid in children with epilepsy: Implications for dose tailoring when switching from oral syrup to sustained-release tablets.

Significant pharmacokinetic (PK) differences exist between different forms of valproic acid (VPA), such as syrup and sustained-release (SR) tablets. This study aimed to develop a population pharmacokinetic (PopPK) model for VPA in children with epilepsy and offer dose adjustment recommendation for switching dosage forms as needed. The study collected 1411 VPA steady-state trough concentrations (Ctrough) from 617 children with epilepsy. Using NONMEM software, a PopPK model was developed, employing a stepwise approach to identify possible variables such as demographic information and concomitant medications. The final model underwent internal and external evaluation via graphical and statistical methods. Moreover, Monte Carlo simulations were used to generate a dose tailoring strategy for typical patients weighting 20-50 kg. As a result, the PK characteristics of VPA were described using a one-compartment model with first-order absorption. The absorption rate constant (ka) was set at 2.64 and 0.46 h-1 for syrup and SR tablets. Body weight and sex were identified as significant factors affecting VPA's pharmacokinetics. The final PopPK model demonstrated acceptable prediction performance and stability during internal and external evaluation. For children taking syrup, a daily dose of 25 mg/kg resulted in the highest probability of achieving the desired target Ctrough, while a dose of 20 mg/kg/day was appropriate for those taking SR tablets. In conclusion, we established a PopPK model for VPA in children with epilepsy to tailor VPA dosage when switching between syrup and SR tablets, aiming to improve plasma VPA concentrations fluctuations.

From "wet" matrices to "dry" blood spot sampling strategy: a versatile LC-MS/MS assay for simultaneous monitoring caffeine and its three primary metabolites in preterm infants.

OBJECTIVES: To update traditional "wet" matrices to dried blood spot (DBS) sampling, based on the liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) technique, and develop a method for simultaneous analyzing caffeine and its three primary metabolites (theobromine, paraxanthine, and theophylline), supporting routine therapeutic drug monitoring (TDM) for preterm infants. METHODS: DBS samples were prepared by a two-step quantitative sampling method, i.e., volumetric sampling of a quantitative 10 µL volume of peripheral blood and an 8 mm diameter whole punch extraction by a methanol/water (80/20, v/v) mixture containing 125 mM formic acid. Four paired stable isotope labeled internal standards and a collision energy defect strategy were applied for the method optimization. The method was fully validated following international guidelines and industrial recommendations on DBS analysis. Cross validation with previously developed plasma method was also proceeded. The validated method was then implemented on the TDM for preterm infants. RESULTS: The two-step quantitative sampling strategy and a high recovery extraction method were developed and optimized. The method validation results were all within the acceptable criteria. Satisfactory parallelism, concordance, and correlation were observed between DBS and plasma concentrations of the four analytes. The method was applied to provide routine TDM services to 20 preterm infants. CONCLUSIONS: A versatile LC-MS/MS platform for simultaneous monitoring caffeine and its three primary metabolites was developed, fully validated, and successfully applied into the routine clinical TDM practices. Sampling method switching from "wet" matrices to "dry" DBS will facilitate and support the precision dosing of caffeine for preterm infants.

Successful LC-MS/MS assay development and validation for determination of valproic acid and its metabolites supporting proactive pharmacovigilance.

Valproic acid (VPA) is a well-documented contributor to liver injury, which is likely caused by the formation of its toxic metabolites. Monitoring VPA and its metabolites is very meaningful for the pharmacovigilance, but the availability of a powerful assay is a prerequisite. In this study, for the first time, a sensitive and specific LC-MS/MS method was developed and validated to simultaneously quantify the concentrations of VPA and its six pestering isomer metabolites (3-OH-VPA, 4-OH-VPA, 5-OH-VPA, 2-PGA, VPA-G, and 2-ene-VPA) in human plasma, using 5-OH-VPA-d7 and VPA-d6 as the internal standards (ISs). We also figured out another tricky problem that the concentrations of the parent drug and the metabolites vary widely. Of note, after protein precipitation and dilution with acetonitrile (ACN) and 50% ACN successively, the analytes and the ISs were successfully separated on a Kinetex C18 column. Intriguingly, sacrificing its signal intensity by elevated collision energy of VPA finally achieved the simultaneous determination. As expected, the method showed great linearity (r > 0.998) over the concentration ranges for all analytes. The inter-day and intra-day accuracy and precision were both acceptable. The method was successfully applied in 127 children with epilepsy. This novel assay will support the VPA-associated pharmacovigilance in the future.

Efficacy and safety of EGFR­TKIs plus Shenqi Fuzheng injection for non-small cell lung cancer patients with EGFR-sensitive mutations.

PURPOSE: The aim of this retrospective study is to evaluate the impact on efficacy and safety between epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) alone and in combination with Shenqi Fuzheng injection (SFI) in patients with advanced NSCLC harboring epidermal growth factor receptor (EGFR) activating mutations. METHODS: Retrospectively, information of 88 patients receiving EGFR-TKIs as first-line targeted treatment or in combination with SFI in the Affiliated Drum Tower Hospital of Nanjing University Medical College and the Affiliated Cancer Hospital of Anhui University of Science and Technology was collected. The primary endpoint was to assess progression-free survival (PFS) and safety of EGFR-TKIs alone or in combination with SFI. RESULTS: Between January 2016 and December 2019, a total of 88 patients were enrolled in this research, including 50 cases in the EGFR-TKIs single agent therapy group and 38 cases in the SFI combined with EGFR-TKIs targeted-therapy group. The median PFS (mPFS) of monotherapy group was 10.50 months (95%CI 9.81-11.19), and 14.30 months (95%CI 10.22-18.38) in the combination therapy group. Compared to the single EGFR-TKIs administration, combinational regimen with SFI exhibited a lower incidence of rash and diarrhea in patients and was even better tolerated. CONCLUSIONS: SFI combined with the first-generation EGFR-TKIs are more efficient, can prominently prolong the PFS and attenuate the adverse reactions in patients with advanced NSCLC with EGFR-sensitive mutations.

Precision caffeine therapy for apnea of prematurity and circadian rhythms: New possibilities open up.

Caffeine is the globally consumed psychoactive substance and the drug of choice for the treatment of apnea of prematurity (AOP), but its therapeutic effects are highly variable among preterm infants. Many of the molecular underpinnings of the marked individual response have remained elusive yet. Interestingly, the significant association between Clock gene polymorphisms and the response to caffeine therapy offers an opportunity to advance our understanding of potential mechanistic pathways. In this review, we delineate the functions and mechanisms of human circadian rhythms. An up-to-date advance of the formation and ontogeny of human circadian rhythms during the perinatal period are concisely discussed. Specially, we summarize and discuss the characteristics of circadian rhythms in preterm infants. Second, we discuss the role of caffeine consumption on the circadian rhythms in animal models and human, especially in neonates and preterm infants. Finally, we postulate how circadian-based therapeutic initiatives could open new possibilities to promote precision caffeine therapy for the AOP management in preterm infants.

Comparison of LC-MS/MS and EMIT methods for the precise determination of blood sirolimus in children with vascular anomalies.

Sirolimus (SRL) is a mammalian target of rapamycin (mTOR) inhibitor. The whole blood concentration of SRL is routinely monitored to tailor dosage and prevent toxicity. Currently, the enzyme multiplied immunoassay technique (EMIT) is often applied to perform therapeutic drug monitoring (TDM) of SRL, but the cross-reactivity with various metabolites is of great concern. A more specific method is required, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, no study on the method comparison of the EMIT and LC-MS/MS for the measurement of whole blood SRL concentration in children with vascular anomalies has been reported. This study developed a simple and sensitive LC-MS/MS assay for the determination of SRL. Meanwhile, consistency between LC-MS/MS and the EMIT was evaluated by linear regression and Bland-Altman analysis. Whole blood samples were deproteinized with methanol for erythrocyte lysis, and the resulting solution was injected into the LC-MS/MS system using the positive electrospray ionization mode. The multiple reaction monitoring transitions of m/z 931.7 → 864.6 and m/z 934.7 → 864.6 were used for SRL and SRL-d3 as the internal standards, respectively. The analytes were separated on a C18 column with a gradient mobile phase (0.1 mM formic acid and 0.05 mM ammonium acetate in methanol/ultrapure water). Blood samples collected from children with vascular anomalies undergoing SRL therapy were tested by EMIT and by LC-MS/MS. The linear range of LC-MS/MS was 0.500-50.0 ng/ml and that of the EMIT was 3.50-30.0 ng/ml. A significant positive correlation between the two assays was established with a regression equation described as [ EMIT ] = 1.281 × [ LC-MS/MS ] + 2.450 (r = 0.8361). Bland-Altman plots showed a mean concentration overestimation of 4.7 ng/ml [95% CI: (-3.1, 12.6)] and a positive bias of 63.1% [95% CI: (-36.1, 162.3)] generated by the EMIT more than that of by LC-MS/MS. In conclusion, the two methods were closely correlated, indicating that switching between the two methods is feasible. Considering the overestimation nature of the EMIT assay, switching from the EMIT to the LC-MS/MS method deserves close attention and necessary re-evaluation for the target therapeutic reference range, may be required when methods are switched within the same clinical laboratory or results are compared between different laboratories.

Population pharmacokinetic modeling of caffeine in preterm infants with apnea of prematurity: New findings from concomitant erythromycin and AHR genetic polymorphisms.

Current standard-dose caffeine therapy results in significant intersubject variability. The aims of this study were to develop and evaluate population pharmacokinetic (PPK) models of caffeine in preterm infants through comprehensive screening of covariates and then to propose model-informed precision dosing of caffeine for this population. A total of 129 caffeine concentrations from 96 premature neonates were incorporated into this study. Comprehensive medical record and genotype data of these neonates were collected for analysis. PPK modeling was performed by a nonlinear mixed effects modeling program (NONMEM). Final models based on the current weight (CW) or body surface area (BSA) were evaluated via multiple graphic and statistical methods. The model-informed dosing regimen was performed through Monte Carlo simulations. In addition to CW or BSA, postnatal age, coadministration with erythromycin (ERY), and aryl hydrocarbon receptor coding gene (AHR) variant (rs2158041) were incorporated into the final PPK models. Multiple evaluation results showed satisfactory prediction performance and stability of the CW- and BSA-based models. Monte Carlo simulations demonstrated that trough concentrations of caffeine in preterm infants would be affected by concomitant ERY therapy and rs2158041 under varying dose regimens. For the first time, ERY and rs2158041 were found to be associated with the clearance of caffeine in premature infants. Similar predictive performance and stability were obtained for both CW- and BSA-based PPK models. These findings provide novel insights into caffeine precision therapy for preterm infants.

Therapeutic drug monitoring of caffeine and its primary metabolites in plasma using LC-ESI-MS/MS for apnea of prematurity treatment: Evaluation of ultrapure water as a surrogate matrix.

The growing evidence has endorsed the view that therapeutic drug monitoring of caffeine for apnea of prematurity is helpful for dose tailoring when the therapeutic response is lacking or toxicity is suspected. However, plasma without caffeine is difficult to obtain. Therefore, a method was developed and validated to measure caffeine and its three primary metabolites (paraxanthine, theobromine and theophylline) using LC-ESI-MS/MS in human plasma and several surrogate matrices. The chromatographic separation of analytes was finally achieved on a Waters Symmetry C18 (4.6 × 75 mm, 3.5 µm) column. Several strategies were successfully applied to overcome the matrix effects: (a) appropriate dilution for sample cleanup; (b) a starting lower proportion of organic phase; and (c) multiple individual stable-labeled isotopic internal standards. The parallelism between the authentic matrix and surrogate matrices was convincing. The recovery of the analytes in both human plasma and rat plasma was acceptable over the linear range (0.500-50.0 µg/ml for caffeine and 0.0100-1.00 µg/ml for three metabolites). The method was successfully applied in 118 samples from 74 preterm infants with apnea of prematurity. The rat plasma or ultrapure water as a surrogate matrix is worthy of recommendation for routine therapeutic drug monitoring of caffeine.

Cerebrospinal fluid metabolic profiling reveals divergent modulation of pentose phosphate pathway by midazolam, propofol and dexmedetomidine in patients with subarachnoid hemorrhage: a cohort study.

BACKGROUND: Agitation is common in subarachnoid hemorrhage (SAH), and sedation with midazolam, propofol and dexmedetomidine is essential in agitation management. Previous research shows the tendency of dexmedetomidine and propofol in improving long-term outcome of SAH patients, whereas midazolam might be detrimental. Brain metabolism derangement after SAH might be interfered by sedatives. However, how sedatives work and whether the drugs interfere with patient outcome by altering cerebral metabolism is unclear, and the comprehensive view of how sedatives regulate brain metabolism remains to be elucidated. METHODS: For cerebrospinal fluid (CSF) and extracellular space of the brain exchange instantly, we performed a cohort study, applying CSF of SAH patients utilizing different sedatives or no sedation to metabolomics. Baseline CSF metabolome was corrected by selecting patients of the same SAH and agitation severity. CSF components were analyzed to identify the most affected metabolic pathways and sensitive biomarkers of each sedative. Markers might represent the outcome of the patients were also investigated. RESULTS: Pentose phosphate pathway was the most significantly interfered (upregulated) pathway in midazolam (p = 0.0000107, impact = 0.35348) and propofol (p = 0.00000000000746, impact = 0.41604) groups. On the contrary, dexmedetomidine decreased levels of sedoheptulose 7-phosphate (p = 0.002) and NADP (p = 0.024), and NADP is the key metabolite and regulator in pentose phosphate pathway. Midazolam additionally augmented purine synthesis (p = 0.00175, impact = 0.13481) and propofol enhanced pyrimidine synthesis (p = 0.000203, impact = 0.20046), whereas dexmedetomidine weakened pyrimidine synthesis (p = 0.000000000594, impact = 0.24922). Reduced guanosine diphosphate (AUC of ROC 0.857, 95%CI 0.617-1, p = 0.00506) was the significant CSF biomarker for midazolam, and uridine diphosphate glucose (AUC of ROC 0.877, 95%CI 0.631-1, p = 0.00980) for propofol, and succinyl-CoA (AUC of ROC 0.923, 95%CI 0.785-1, p = 0.000810) plus adenosine triphosphate (AUC of ROC 0.908, 95%CI 0.6921, p = 0.00315) for dexmedetomidine. Down-regulated CSF succinyl-CoA was also associated with favorable outcome (AUC of ROC 0.708, 95% CI: 0.524-0.865, p = 0.029333). CONCLUSION: Pentose phosphate pathway was a crucial target for sedatives which alter brain metabolism. Midazolam and propofol enhanced the pentose phosphate pathway and nucleotide synthesis in poor-grade SAH patients, as presented in the CSF. The situation of dexmedetomidine was the opposite. The divergent modulation of cerebral metabolism might further explain sedative pharmacology and how sedatives affect the outcome of SAH patients.

PPARα agonist fenofibrate relieves acquired resistance to gefitinib in non-small cell lung cancer by promoting apoptosis via PPARα/AMPK/AKT/FoxO1 pathway.

Recent studies show that intracellular accumulation of cholesterol leads to acquired resistance to gefitinib in non-small cell lung cancer (NSCLC) cells. In this study we investigated how to regulate the cholesterol levels in gefitinib-resistant NSCLC cells. We showed that intracellular cholesterol levels in gefitinib-resistant cell lines (PC-9/GR, H1975, H1650, and A549) were significantly higher than that in gefitinib-sensitive cell line (PC-9). Treatment with gefitinib (5 µM) significantly increased intracellular cholesterol levels in PC-9/GR, H1975, and H1650 cells. Gefitinib treatment downregulated the expression of PPARα, LXRα, and ABCA1, leading to dysregulation of cholesterol efflux pathway. We found that a lipid-lowering drug fenofibrate (20, 40 µM) dose-dependently increased the expression of PPARα, LXRα, and ABCA1, decreased the intracellular cholesterol levels, and enhanced the antiproliferative effects of gefitinib in PC-9/GR, H1975, and H1650 cells. We revealed that fenofibrate increased the gefitinib-induced apoptosis via regulating the key proteins involved in the intrinsic apoptosis pathway. In PC-9/GR, H1975 and H1650 cells, fenofibrate dose-dependently increased the expression of AMPK, FoxO1, and decreased the expression of AKT, which were remarkably weakened by knockdown of PPARα. In PC-9/GR cell xenograft mice, combined administration of gefitinib (25 mg · kg-1 · d-1) and fenofibrate (100 mg · kg-1 · d-1) caused remarkable inhibition on tumor growth as compared to treatment with either drug alone. All the results suggest that fenofibrate relieves acquired resistance to gefitinib in NSCLC by promoting apoptosis via regulating PPARα/AMPK/AKT/FoxO1 pathway. We propose that combination of gefitinib and fenofibrate is a potential strategy for overcoming the gefitinib resistance in NSCLC.

Switching Between LC-ESI-MS/MS and EMIT Methods for Routine TDM of Valproic Acid in Pediatric Patients With Epilepsy: What Clinicians and Researchers Need to Know.

Background: Valproic acid (VPA) is a widely used antiseizure medication and its dosing needs to be tailored individually through therapeutic drug monitoring (TDM) to avoid or prevent toxicity. Currently, immune-enzymatic assays such as Enzyme Multiplied Immunoassay Technique (EMIT), and Liquid Chromatography (LC)-based techniques, particularly coupled to Electrospray Ionization Tandem Mass Spectrometry (LC-ESI-MS/MS), resulting a potential lack of concordance between laboratories. Methods: In this study, plasma VPA concentrations were determined for 711 pediatric patients with epilepsy by a routine EMIT assay and by a validated in-house LC-ESI-MS/MS method on the same group of samples, aimed to address the aforementioned concern. Consistency between two assays was evaluated using linear regression and Bland-Altman analysis. Results: The calibration curve was linear in the range of 5.00-300 µg/ml for LC-ESI-MS/MS method and 1.00-150 µg/ml for EMIT assay, respectively. The two methods were proven to be accurate with quality control samples. As a result, a significant correlation between two methods was obtained with a regression equation described as [ EMIT ] = 1.214 × [ LC - ESI - MS / MS ] + 3.054 (r 2 = 0.9281). Bland-Altman plot showed a mean bias of 14.5 µg/ml (95% confidence interval (CI) (-0.2, 29.2) and a mean increase of 27.8% (95% CI (3.3, 52.4) measured by EMIT assay more than that measured by LC-ESI-MS/MS method. Conclusion: In conclusion, two methods were closely correlated, but EMIT assay overestimate VPA levels in human plasma compared with LC-ESI-MS/MS method. Due to the observed significant discordance between the tested methods, switching from immunoassays to LC-based techniques for TDM of VPA deserves close attention and therapeutic range of 35.0-75.0 µg/ml may be feasible. However, further studies are needed to evaluate the eligibility of this alternative range in the clinical practice. Clinicians should be informed when switching the VPA quantitation methods during the clinical practice.

Caffeine for the Pharmacological Treatment of Apnea of Prematurity in the NICU: Dose Selection Conundrum, Therapeutic Drug Monitoring and Genetic Factors.

Caffeine citrate is the drug of choice for the pharmacological treatment of apnea of prematurity. Factors such as maturity and genetic variation contribute to the interindividual variability in the clinical response to caffeine therapy in preterm infants, making the optimal dose administered controversial. Moreover, the necessity for therapeutic drug monitoring (TDM) of caffeine is still worth discussing due to the need to achieve the desired target concentrations as well as concerns about the safety of higher doses. Therefore, we reviewed the pharmacokinetic profile of caffeine in preterm infants, evidence of the safety and efficacy of different doses of caffeine, therapeutic concentration ranges of caffeine and impact of genetic variability on caffeine therapy. Whereas the safety and efficacy of standard-dose caffeine have been demonstrated, evidence for the safety of higher administered doses is insufficient. Thus, preterm infants who lack clinical response to standard-dose caffeine therapy are of interest for TDM when dose optimization is performed. Polymorphisms in pharmacodynamics-related genes, but not in pharmacokinetics-related genes, have a significant impact on the interindividual variability in clinical response to caffeine therapy. For preterm infants lacking clinical response, how to develop individualized medication regimens for caffeine remains to be explored.

Identification and validation of inferior prognostic genes associated with immune signatures and chemotherapy outcome in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a group of heterogeneous hematological malignancies. We identified key genes as ITGAM and lncRNA ITGB2-AS1 through different bioinformatics tools. Furthermore, qPCR was performed to verify the expression level of essential genes in clinical samples. Retrospective research on 179 AML cases was used to investigate the relationship between the expression of ITGAM and the characteristics of AML. The critical gene relationship with immune infiltration in AML was estimated. The clinical validation and prognostic investigation showed that ITGAM, PPBP, and ITGB2-AS1 are highly expressed in AML (P < 0.001) and significantly associated with the overall survival in AML. Moreover, the retrospective research on 179 clinical cases showed that positive expression of ITGAM is substantially related to AML classification (P < 0.001), higher count of white blood cells (P < 0.01), and poor chemotherapy outcome (P < 0.05). Furthermore, based on grouping ITGAM as the high and low expression in TCGA-LAML profile, we found that genes in the highly expressed ITGAM group are mainly involved in immune infiltration and inflammation-related signaling pathways. Finally, we discovered that the expression level of ITGAM and lncRNA ITGB2-AS1 are not just closely related to the immune score and stromal score (P < 0.001) but also significantly positively correlated with various Immune signatures in AML (P < 0.001), indicating the association of these genes with immunosuppression in AML. The prediction of candidate drugs indicated that certain immunosuppressive drugs have potential therapeutic effects for AML. The critical genes could be used as potential biomarkers to evaluate the survival and prognosis of AML.

Identification of MiR-93-5p Targeted Pathogenic Markers in Acute Myeloid Leukemia through Integrative Bioinformatics Analysis and Clinical Validation.

Acute myeloid leukemia (AML) is a type of hematological malignancy with diverse genetic pathogenesis. Identification of the miR-93-5p targeted pathogenic markers could be useful for AML diagnosis and potential therapy. We collected 751 miR-93-5p targeted and AML-related genes by integrating the results of multiple databases and then used the expression profile of TCGA-LAML to construct a coexpression function network of AML WGCNA. Based on the clinical phenotype and module trait relationship, we identified two modules (brown and yellow) as interesting dysfunction modules, which have a significant association with cytogenetics risk and FAB classification systems. GO enrichment and KEGG analysis showed that these modules are mainly involved with cancer-associated pathways, including MAPK signal pathway, p53 signal pathway, JAK-STAT signal pathway, TGF-beta signaling pathway, mTOR signaling pathway, VEGF signaling pathway, both associated with the occurrence of AML. Besides, using the STRING database, we discovered the top 10 hub genes in each module, including MAPK1, ACTB, RAC1, GRB2, MDM2, ACTR2, IGF1R, CDKN1A, YWHAZ, and YWHAB in the brown module and VEGFA, FGF2, CCND1, FOXO3, IGFBP3, GSF1, IGF2, SLC2A4, PDGFBM, and PIK3R2 in the yellow module. The prognosis analysis result showed that six key pathogens have significantly affected the overall survival and prognosis in AML. Interestingly, VEGF with the most significant regulatory relationship in the yellow modules significantly positively correlated with the clinical phenotype of AML. We used qPCR and ELISA to verify miR-93-5p and VEGF expression in our clinical samples. The results exhibited that miR-93-5p and VEGF were both highly expressed in AML.

MiR-21-3p triggers cardiac fibroblasts pyroptosis in diabetic cardiac fibrosis via inhibiting androgen receptor.

AIMS/HYPOTHESIS: MicroRNA-21 has been implicated in diabetic complication, including diabetic cardiomyopathy. However, there is limited information regarding the biological role of the miR-21 passenger strand (miR-21-3p) in diabetic cardiac fibrosis. The aim of this study was to investigate the role of miR-21-3p and its target androgen receptor in STZ-induced diabetic cardiac fibrosis. METHODS: The pathological changes and collagen depositions was analyzed by HE, Sirius Red staining and Masson's Trichrome Staining. MiR-21-3p, AR, NLRP3, caspase1 and collagen I expression were analyzed by western blotting, immunohistochemistry, immunofluorescence, qRT-PCR, miR one step qRT-PCR, respectively. A luciferase reporter assay was used to verify the interaction between miR-21 and the 3' untranslated region (3'UTR) of AR. RESULTS: Our results indicated that miR-21-3p level was up-regulated, while AR was decreased in STZ-induced diabetic cardiac fibrosis tissues and cardiac fibroblast. High glucose triggers cardiac fibroblasts pyroptosis and collagen deposition. Gain-of-function and loss-of-function assays demonstrated that miR-21-3p mediated the crucial role in diabetic cardiac fibrosis. Our results show that miR-21-3p bound to the 3'UTR of AR post-transcriptionally repressed its expression. We also found AR, which regulates cardiac fibroblasts pyroptosis and collagen deposition through caspase1 signaling. CONCLUSIONS: /interpretation: Taken together, our study showed that miR-21-3p aggravates STZ-induced diabetic cardiac fibrosis through the caspase1 pathways by suppressing AR expression.

Caffeine Therapy for Apnea of Prematurity: Role of the Circadian CLOCK Gene Polymorphism.

Standard-dose caffeine citrate has been routinely prescribed for apnea of prematurity (AOP) management; however, some preterm infants respond well to the therapy while others do not. The AOP phenotype has been attributed solely to the immature control of the respiratory system consequent to preterm birth, but there are also important genetic influences. Based on our previous report, we tested the hypothesis that the human circadian locomotor output cycles kaput (CLOCK) gene polymorphisms play a role in the response to caffeine citrate therapy in preterm infants. We also studied the interactions of the circadian clock with aryl hydrocarbon receptor (AHR) signaling pathways in preterm babies who received caffeine citrate. This single-center study collected data from 112 preterm infants (<35 weeks gestational age) between July 2017 and July 2018, including apnea-free (n = 48) and apneic (n = 64) groups. Eighty-eight candidate single nucleotide polymorphisms (SNPs) were tested using the MassARRAY system. Association analysis was performed using the PLINK Whole Genome Data Analysis Toolset and SNPStats software. Linkage disequilibrium (LD) and haplotype analyses were performed using Hapview software. No significant intergroup differences in allele distributions or genotype frequencies of CYP1A2, CYP3A4, CYP3A5, and CYP3A7 were detected in our study on preterm babies. Two more SNPs in AHR were found to be associated with determining the response to caffeine citrate therapy in our pediatric patients. Of the 46 candidate SNPs in the CLOCK gene, 26 were found to be associated with determining the response to caffeine treatment in these babies. Interestingly, a significant association was retained for 18 SNPs in the CLOCK gene after false discovery rate correction. Moreover, strong LD formed in those variants in AHR, ADORA2A, and CLOCK genes was confirmed to be significantly associated with a better response to standard-dose caffeine therapy. In summary, CLOCK gene polymorphisms play a role in determining the response to caffeine therapy in premature neonates with AOP. However, whether the AHR and CLOCK signaling pathways crosstalk with each other during caffeine treatment remains largely unclear. Future clinical studies including more immature babies and basic research are needed to explore the mechanism by which circadian rhythms affect the response to caffeine therapy.

DNMT1 deregulation of SOCS3 axis drives cardiac fibroblast activation in diabetic cardiac fibrosis.

Cardiac fibrosis is one of the main pathological manifestations of diabetic cardiomyopathy (DCM). Cardiac fibroblast activation is a key effector of cells resulting in diabetic cardiac fibrosis. However, the underlying mechanism of cardiac fibroblast activation and diabetic cardiac fibrosis remains unclear. Accumulating evidence suggests that DNA methylation alterations play a central role in cardiac fibroblast activation. In this study, we demonstrated that DNA methyltransferase 1 (DNMT1)-mediated suppression of cytokine signaling 3 (SOCS3) promoter hypermethylation leads to downregulation of SOCS3 expression in diabetic cardiac fibrosis. High glucose-induced expression of DNMT1 was increased in cardiac fibroblasts, while the expression of SOCS3 was decreased. Downregulation of SOCS3 facilitated activation of STAT3 to promote cardiac fibroblast activation and collagen deposition. Genetic or pharmacological inactivation of DNMT1 reversed the activated phenotype of cardiac fibroblasts. Clinically, we observed a significant inverse correlation between DNMT1 and SOCS3 expression levels, and loss of SOCS3 expression or increased expression of DNMT1. Taken together, these findings identify DNMT1 silencing of SOCS3 axis as a driver of cardiac fibroblast activation in diabetic cardiac fibrosis. These results provide a scientific and new explanation of the underlying mechanism of diabetic cardiac fibrosis.

DNA methyltransferase-1 inactivation of androgen receptor axis triggers homocysteine induced cardiac fibroblast autophagy in diabetic cardiac fibrosis.

Diabetic cardiac fibrosis is one of the main pathological manifestations of diabetic cardiomyopathy (DCM). Cardiac fibroblast autophagy plays critical roles in diabetic cardiac fibrosis, however, the underlying mechanism of cardiac fibroblast autophagy and diabetic cardiac fibrosis still largely unknown. The aim of the study was to investigate the mechanism of DNMT1 mediated DNA methylation alterations control cardiac fibroblast autophagy in diabetic cardiac fibrosis. We employed streptozotocin (STZ)-induced rats DCM, DCM patient and Hcy induced cardiac fibroblast autophagy. Heart tissue sections were stained with H&E, Sirius Red and Masson's trichrome stain. The expression of DNMT1, AR, Collagen genes mRNA was detected by qRT-PCR. MSP and BSP detected the methylation status of the AR promoter. The expression of DNMT1, AR, Collagen and autophagy-related proteins were detected by Western blotting, Immunofluorescence, Immunohistochemistry. Gain and loss function of AR and DNMT1 in cardiac fibroblast was analyzed. DNMT1 inhibition or knockdown elevated the expression of AR in cardiac fibroblast. Furthermore, we found that AR negatively regulation of Hcy induced cardiac fibroblast autophagy. We demonstrated that DNMT1 enhances cardiac fibroblast autophagy in diabetic cardiac fibrosis through inhibiting AR axis. In conclusion, our results provide new insight into the DNMT1 inactivation of AR axis triggers cardiac fibroblast autophagy in diabetic cardiac fibrosis.

MeCP2 inactivation of LncRNA GAS5 triggers cardiac fibroblasts activation in cardiac fibrosis.

Long non coding RNA growth arrest-specific transcript 5 (LncRNA GAS5) participate in the formation of fibrosis diseases. However, the key role of LncRNA GAS5 in the development of cardiac fibrosis remains unclear. Accumulating evidence suggests that DNA methylation alterations play a central role in cardiac fibroblast activation. In this study, we explored MeCP2 inactivation of LncRNA GAS5 leads to down regulation of LncRNA GAS5 expression in cardiac fibrosis. Gain and loss function of LncRNA GAS5 and MeCP2 was analyzed. The expression of LncRNA GAS5 was significantly decreased in cardiac fibrosis tissues, while MeCP2 was significantly increased. Moreover, the expression of MeCP2 was increased in TGF-ß1 induced cardiac fibroblasts, while the expression of LncRNA GAS5 was decreased. Down regulation of LncRNA GAS5 resulted in increasing cellular proliferation. In contrast, exogenous over expression of LncRNA GAS5 in cardiac fibroblasts inhibited cell proliferation. 5-AzadC or knockdown of MeCP2 treatment significantly restored LncRNA GAS5 expression in cardiac fibroblasts, while over expression of MeCP2 treatment significantly inhibited LncRNA GAS5 expression in cardiac fibroblasts. In summary, these results suggested that MeCP2 silencing of LncRNA GAS5 triggers cardiac fibroblasts activation in cardiac fibrosis.