BMP-4 impedes endothelial cell migration in neointimal hyperplasia via FoXO-3 specific modulation of reactive oxygen species.

BACKGROUND AND AIMS: Endothelial cell injury causes vascular barrier dysfunction and leukocyte recruitment to the underlying tissue. Bone morphogenetic protein 4 (BMP-4) is a transforming growth factor that exerts pro-inflammatory effects on the endothelium. Here, we investigated the effects of BMP-4 on endothelial cell (EC) migration following balloon injury in SD rats. METHODS: An intimal hyperplasia model was established using balloon injury. Hematoxylin-eosin staining (HE) and silver staining were used to detect the alteration of endothelial cells recovery after balloon injury. Serum BMP-4 levels were assessed by ELISA. Human umbilical vein endothelial cells (HUVECs) were cultured. MTT assay was used to measure cell viability. Protein expression was detected by Western blot. Intracellular reactive oxygen species (ROS) was detected by dichloro-dihydro-fluorescein diacetate (DCFH-DA). HUVECs migration was measured via transwell assay and scratch wound assay. RESULTS: The results indicated that BMP-4 inhibition significantly decreased total plasma activity of BMP-4 and reduced neointimal hyperplasia by stimulating endothelial cell migration, but did not affect the medial area following balloon injury. BMP-4 suppressed the formation of ROS via forkhead box O3 (FoXO-3)/superoxide dismutase 1 (SOD-1). In vitro, a high level of ROS induced by BMP-4 impeded HUVECs migration. CONCLUSIONS: The results suggest that BMP-4 inhibition is a potential means of preventing intimal hyperplasia formation after balloon injury.

Association of FOXO3 Blood DNA Methylation with Cancer Risk, Cancer Survival, and Mortality.

Genetic variants in FOXO3 are associated with longevity. Here, we assessed whether blood DNA methylation at FOXO3 was associated with cancer risk, survival, and mortality. We used data from eight prospective case-control studies of breast (n = 409 cases), colorectal (n = 835), gastric (n = 170), kidney (n = 143), lung (n = 332), prostate (n = 869), and urothelial (n = 428) cancer and B-cell lymphoma (n = 438). Case-control pairs were matched on age, sex, country of birth, and smoking (lung cancer study). Conditional logistic regression was used to assess associations between cancer risk and methylation at 45 CpGs of FOXO3 included on the HumanMethylation450 assay. Mixed-effects Cox models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations with cancer survival (total n = 2286 deaths). Additionally, using data from 1088 older participants, we assessed associations of FOXO3 methylation with overall and cause-specific mortality (n = 354 deaths). Methylation at a CpG in the first exon region of FOXO3 (6:108882981) was associated with gastric cancer survival (HR = 2.39, 95% CI: 1.60-3.56, p = 1.9 × 10-5). Methylation at three CpGs in TSS1500 and gene body was associated with lung cancer survival (p < 6.1 × 10-5). We found no evidence of associations of FOXO3 methylation with cancer risk and mortality. Our findings may contribute to understanding the implication of FOXO3 in longevity.

The DNA methylation of FOXO3 and TP53 as a blood biomarker of late-onset asthma.

BACKGROUND: Late-onset asthma (LOA) is beginning to account for an increasing proportion of asthma patients, which is often underdiagnosed in the elderly. Studies on the possible relations between aging-related genes and LOA contribute to the diagnosis and treatment of LOA. Forkhead Box O3 (FOXO3) and TP53 are two classic aging-related genes. DNA methylation varies greatly with age which may play an important role in the pathogenesis of LOA. We supposed that the differentially methylated sites of FOXO3 and TP53 associated with clinical phenotypes of LOA may be useful biomarkers for the early screening of LOA. METHODS: The mRNA expression and DNA methylation of FOXO3 and TP53 in peripheral blood of 43 LOA patients (15 mild LOA, 15 moderate LOA and 13 severe LOA) and 60 healthy controls (HCs) were determined. The association of methylated sites with age was assessed by Cox regression to control the potential confounders. Then, the correlation between differentially methylated sites (DMSs; p-value < 0.05) and clinical lung function in LOA patients was evaluated. Next, candidate DMSs combining with age were evaluated to predict LOA by receiver operating characteristic (ROC) analysis and principal components analysis (PCA). Finally, HDM-stressed asthma model was constructed, and DNA methylation inhibitor 5-Aza-2'-deoxycytidine (5-AZA) were used to determine the regulation of DNA methylation on the expression of FOXO3 and TP53. RESULTS: Compared with HCs, the mRNA expression and DNA methylation of FOXO3 and TP53 vary significantly in LOA patients. Besides, 8 DMSs from LOA patients were identified. Two of the DMSs, chr6:108882977 (FOXO3) and chr17:7591672 (TP53), were associated with the severity of LOA. The combination of the two DMSs and age could predict LOA with high accuracy (AUC values = 0.924). In HDM-stressed asthma model, DNA demethylation increased the expression of FOXO3 and P53. CONCLUSIONS: The mRNA expression of FOXO3 and TP53 varies significantly in peripheral blood of LOA patients, which may be due to the regulation of DNA methylation. FOXO3 and TP53 methylation is a suitable blood biomarker to predict LOA, which may be useful targets for the risk diagnosis and clinical management of LOA.

Novel association between FOXO3 rs2232365 polymorphism and late-onset preeclampsia: a case-control candidate genetic study.

BACKGROUND: Both genetic susceptibility and dysregulated lipid metabolism are important susceptibilities to preeclampsia. In the study, we devote to investigate the associations of FOXO3 and TLR7 genetic polymorphisms with preeclampsia in a Chinese population. METHODS: This case-control study involved 335 Han Chinese pregnant women, including 177 pregnant women with preeclampsia and 158 healthy controls. The preeclampsia group was further sub-grouped into early-onset preeclampsia (EOPE, n = 70)and late-onset preeclampsia (LOPE, n = 107. Three single nucleotide polymorphisms (SNPs), including FOXO3 (rs2232365, rs3761548), and TLR7 rs3853839 were genotyped by multiplex PCR for targeted next-generation sequencing. The χ2 test and multiple interaction effect analyses were performed to determine the association of three SNPs with serum lipid levels and thyroid function in women with preeclampsia. RESULTS: The genotype (CC vs. TT + CT) distribution of rs2232365 revealed a significant association with LOPE (P = 0.004, odds ratio = 3.525 (0.95 CI: 1.498-8.164)). No significant difference was found in the genotype and allele frequencies of rs3761548 and rs3853839 between controls and cases (P > 0.05). Moreover, the genotype CT/TT of rs2232365 was significantly correlated with increased TG/HDL levels in the LOPE group (p = 0.014). CONCLUSIONS: The polymorphisms of rs2232365 are associated with the risk of LOPE and may modulate TG/HDL levels in pregnant women with LOPE.

Serum FOXO3A: A ray of hope for early diagnosis of Alzheimer's disease.

Diagnosis of Alzheimer's disease (AD) is often difficult because of distinct and subjective clinical features, especially in the early stage. FOXO3a protein present in the cognitive centre of brain in inferior temporal region and parahippocampus. FOXO3a can be a potential novel target against AD. AD, Mild Cognitive impairment (MCI) and Geriatric Control (GC) were recruited after diagnosis by clinical assessment, MRI, TauPET and FDG-PET. We have quantified serum FOXO3a by surface plasmon resonance (SPR) and compare with TauPET between of AD, MCI patients and GC. Serum FOXO3A was significantly lower in AD (1.42 ± 0.09 ng/µl) compare to MCI (1.61 ± 0.14 ng/µl) and GC (1.89 ± 0.07 ng/µl). However, the Tau was higher in AD both in serum and also in PET scan. Serum pTau was significantly over-expressed in AD (0.176 ± 0.03 ng/µl), compare to other groups; MCI (0.16 ± 0.014 ng/µl) and GC (0.15 ± 0.024 ng/µl). Serum FOXO3A could significantly differentiate AD vs MCI, MCI vs GC and AD vs GC. However, Tau protein could only differentiate AD vs GC but not MCI vs GC. Serum FOXO3A may serve as novel blood marker for early detection for AD and target for therapeutic intervention.

Longevity and healthy ageing genes FOXO3A and SIRT3: Serum protein marker and new road map to burst oxidative stress by Withania somnifera.

BACKGROUND: Ageing process is characterized by a decline in function; different age related diseases and excessive age associated mortality. There has always been a quest for easily accessible biomarkers to monitor and identify the development of age-associated stress for providing new anti-ageing strategies. Forkhead box protein O3A (FOXO3A) and Sirtuin3 (SIRT3) are such potential markers which plays important role in a wide variety of cellular mechanisms and has been proposed to be an ideal candidate to study longevity and are potential candidate for healthy ageing by oxidative burst. OBJECTIVES: In this study we quantified FOXO3A and SIRT3 proteins in human serum with increasing age and in-vitro assessment of modulation of their expression by the treatment of Withania somnifera (Ashwagandha). METHODOLOGY: Four hundred seventy three subjects were enrolled for the study and were divided into three groups according to increasing age [20-30years (young), 60-79years (old) and ≥80years (oldest)]. Serum levels of FOXO3A and SIRT3 proteins were estimated by Surface Plasmon Resonance (SPR) and validated by ELISA and Western blot. The statistical analysis was done with student's unpaired t-test, one way ANOVA by Stata9 and Graph pad prism5. The expression of these proteins were also analysed in stress induced HEK-293 cell line and level was observed by treatment with stress releasing compound Ashwagandha. RESULTS: In this cross sectional observational study, the serum concentration of FOXO3A and SIRT3 declined significantly (p<0.0001) with increasing age and even after adjustment with all geriatric co-morbidities the level remain downregulated with age. In the stress inducible cell line showed reduced level of proteins which gets upregulated by the treatment of Ashwagandha. CONCLUSION: This is the first report of inverse relation of age with human serum FOXO3A and SIRT3 and can be excellent marker for ageing with good therapeutic importance for maintaining healthy ageing.