Targeting prolyl-isomerase Pin1 prevents mitochondrial oxidative stress and vascular dysfunction: insights in patients with diabetes

The present study demonstrates that Pin1 is a common activator of key pathways involved in diabetic vascular disease in different experimental settings including primary human endothelial cells, knockout mice, and diabetic patients. Gene silencing and genetic disruption of Pin1 prevent hyperglycaemia-induced mitochondrial oxidative stress, endothelial dysfunction, and vascular inflammation. Moreover, we have translated our findings to diabetic patients. In line with our experimental observations, Pin1 up-regulation is associated with impaired flow-mediated dilation, increased oxidative stress, and plasma levels of adhesion molecules. In perspective, these findings may provide the rationale for mechanism-based therapeutic strategies in patients with diabete

Targeting Prolyl-isomerase-1 protects against hyperglycemia-induced endothelial dysfunction and vascular inflammation: alterations in patients with type 2 diabetes

Introduction: Prolyl-isomerase-1 (Pin1) regulates function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. Recent studies showed that Pin1 favours cancerogenesis through reactive oxygen species (ROS) production and inflammation. Whether Pin1 partecipates to vascular disease is unknown. Hypothesis: This study investigates the role of Pin1 in diabetes-related vascular dysfunction. Methods: Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5mmol/L) or high glucose concentrations (HG, 25 mmol/L) in the presence or in the absence of Pin1 pharmacological inhibitor Juglone or siRNA-mediated knockdown. Diabetes was induced by streptozotocin in C57/B6 mice and animals treated with Pin1 siRNA i.v or Juglone i.p for 30 days. Endothelial function was assessed by dose-response curve with acetylcoline (10-9-10-6 mol/L). Mitochondrial ROS were measured by ESR spectroscopy. Pin1 gene expression was assessed in peripheral blood monocytes (PBM) of 37 patients with type 2 diabetes (T2DM) and 20 age-matched controls and correlated with flow-mediated vasodilation (FMD) of the brachial artery, urinary oxidative marker 8-isoprostaglandinF2α (8-isoPGF2α) and plasma adhesion molecules VCAM-1, ICAM-1 and MCP-1. Results: Pin1 expression increased in HAECs exposed to HG (289±22% vs. NG, p<0.01) and aortas of diabetic mice (216±32% vs. controls, p<0.05). Immunoprecipitation showed that Pin1 recognizes phosphoserine motifs of the pro-oxidant mitochondrial adaptor p66Shc and NF-kB p65. Interestingly, Juglone or Pin1siRNA prevented p66Shc-induced ROS production and suppressed NF-kB-dependent upregulation of adhesion molecules. In diabetic mice, silencing of Pin1 or Juglone prevented endothelial dysfunction, ROS production and vascular inflammation. Of note, Pin1 mRNA was significantly upregulated in PBM of T2DM patients as compared with controls (370±97 vs. 25±28,p<0.01) and correlated with FMD (r=-0.33, p<0.01), urinary 8-isoPGF2α (r=0.46, p<0.05), VCAM-1 (r=0.58, p<0.05) and ICAM-1 (r=0.55, p<0.05). Conclusions: Pin1 may critically participate to vascular disease in T2DM patients

Epigenetic remodeling of Nox2 by acetyltransferase Gcn5 drives hyperglycemia-induced endothelial oxidative stress

Introduction: Epigenetic modifications are recently emerging as important modulators of gene expression. The mammalian acetyltransferase gene non-derepressible 5 (GCN5) causes Nox2 upregulation in neutrophils through acetylation of histones. Whether GCN5 modulates oxidative stress in the cardiovascular system remains largely unknown. Hypothesis: The present study investigates GCN5 role in the modulation of diabetes-related endothelial oxidative stress. Methods: Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5 mmol/L) or high glucose concentrations (HG, 25 mmol/L) in the presence or in the absence of GCN5 pharmacological inhibitor CPTH2 or siRNA-mediated knockdown. Superoxide anion (O2-) was measured by ESR spectroscopy. Chromatin immunoprecipitation (ChIP) was performed to investigate epigenetic modifications on Nox2 promoter induced by GCN5. In parallel, 30 patients with type 2 diabetes (T2DM) and 18 age-matched healthy controls were enrolled. All patients underwent flow-mediated vasodilation (FMD) of the brachial artery to assess endothelial function. Urinary levels of 8-isoprostaglandinF2α (8-isoPGF2α) were measured as a marker of oxidative stress. Gene expression of GCN5 and Nox2 was assessed from peripheral blood monocytes (PBM). Data are presented as fold change (FC). Results: HG caused upregulation of NADPH subunit Nox2 (FC vs. NG: 2.5, p<0.05). Interestingly, GCN5 inhibition or siRNA prevented hyperglycemia-induced Nox2 overexpression and subsequent O2- increase. ChIP assay showed that GCN5 binds human Nox2 promoter and mediates lysine 14 acetylation of histone 3, leading to an open chromatin and active transcription. GCN5 expression was increased in PBM of T2DM patients (FC vs. NG: 3.04, p<0.05) and significantly correlated with Nox2 transcript (r=0.32, p<0.05). Consistently, GCN5 upregulation paralleled endothelial dysfunction (r=-0.33, p<0.05) and oxidative stress (r=0.46, p<0.05), as assessed by FMD and 8-isoPGF2α, respectively. Conclusions: Epigenetic changes operated by GCN5 cause endothelial upregulation of Nox2 and subsequent oxidative stress in diabetes. Our findings provide a novel therapeutic opportunity for the prevention of diabetes-related vascular oxidative stress

Epigenetic signatures of p66Shc promoter contribute to persistent endothelial dysfunction in type 2 diabetics with optimal glycemic control

Introduction: optimal glycemic control (OGC) has failed to improve cardiovascular outcomes in patients with diabetes. We have recently reported that epigenetic regulation of the mitochondrial adaptor p66Shc, critically involved in oxidative stress, accounts for persistent endothelial dysfunction in diabetic mice with OGC. Hypothesis: we investigate whether epigenetic changes of p66Shc contribute to residual oxidative stress and vascular disease in patients with type 2 diabetes (T2DM). Methods: 7 patients with newly-diagnosed T2DM and 7 age-matched controls were studied (age 46±3 vs. 42±7 years, p=NS). After the enrolment, T2DM patients were assigned to OGC for 6 months. Glycated haemoglobin (HbA1c) and continuous blood glucose monitoring (CGM) were used as markers of glycemic control. At baseline and follow-up, patients underwent flow-mediated vasodilation (FMD) of the brachial artery. Urinary levels of 8-isoprostaglandinF2α (8-isoPGF2α) were measured as a marker of oxidative stress. p66Shc mRNA expression was assessed from peripheral blood monocytes (PBM). Chromatin immunoprecipitation (ChIP) and real time PCR were performed to investigate histone acetylation and DNA methylation of p66Shc promoter, respectively. Results: Hb1Ac (9.4±2 vs. 6.9±1%, p<0.01) and CGM (235±25 vs. 131±28 mg/dl, p<0.05) confirmed OGC in T2DM patients. As compared with controls. T2DM patients showed blunted FMD (6.8±1.9 vs. 8.6±1.4 %, p<0.05), increased urinary 8-isoPGF2α levels (295±100 vs. 33±9 pg/mL, p<0.05) and p66Shc gene upregulation (0.18±0.06 vs.0.05±0.03 AU, p<0.05). However, OGC did not rescue endothelial function (FMD 6.9±1.7 vs. 6.8±1.9%, p=NS), oxidative stress (295±100 vs. 292±85 pg/ml, p=NS) and p66Shc upregulation (0.18±0.06 vs.0.22±0.1, AU, p=NS). T2DM patients showed lysine 14 acetylation of histone 3 binding p66Shc promoter and hypomethylation of CpG dinucleotides, two critical epigenetic markers favouring p66Shc overexpression. Interestingly, these epigenetic changes remained despite OGC and correlated with persistent endothelial dysfunction and oxidative stress. Conclusions: Epigenetic regulation of p66Shc gene may contribute to the residual burden of vascular disease in T2DM individuals with OGC

Genetic deletion of p66Sh protects against obesity-induced mitochondrial dysfunction, oxidative stress and vascular inflammation

Introduction: Leptin deficiency in mice (LepOb/Ob) triggers high caloric intake leading to obesity/insulin resistance and activates several reactive oxygen species (ROS) generating pathways. The mitochondrial adaptor p66Shc is a crucial player in ROS-mediated vascular damage. Hypothesis: The present study investigates p66Shc role in this model. Methods: Three groups of male mice were studied (C57/B6, 4-6 months old, n=10/group): 1) wild-type (WT), 2)leptin-deficient (LepOb/Ob), 3) double-mutant (p66Shc-/--LepOb/Ob). Plasma 8-iso-prostaglandin-F2α (8-iso-PGF2α) was measured as a marker of oxidative stress. mRNA and protein expression were assessed by real-time PCR and immunoblotting. ESR spectroscopy assessment of superoxide anion (O2-) levels and mitochondrial swelling were performed in mitochondria isolated from mouse aortas. The interaction between p66Shc and cytochrome c was investigated by pull-down assays. Organ chamber experiments were performed to assess endothelium-dependent relaxations to acetylcholine (Ach, 10-9-10-6 mol/L). NF-kB activity was determined as p65 nuclear translocation. Results: Plasma 8-iso-PGF2α as well as aortic O2- levels were significantly higher in LepOb/Ob than WT. This increase was abolished in p66Shc-/--LepOb/Ob mice. Oxidative stress in LepOb/Ob mice was associated with p66Shc upregulation, mitochondrial translocation and swelling. Consistently, co-immunoprecipitation of p66Shc with cytochrome c and impaired Ach-induced relaxations were observed in the aortas of LepOb/Ob but not in WT mice. By contrast, in p66Shc-/--LepOb/Ob mice endothelial dysfunction and mitochondrial swelling did not occur. In agreement with these findings, a preserved activation of Akt/eNOS pathway was also observed. Genetic disruption of p66Shc in obese mice prevented vascular activation of NF-kB signalling and upregulation of VCAM-1, ICAM-1, IL-6 and MCP-1. Conclusions: Our results suggest that p66Shc is critically involved in obesity-induced oxidative stress, endothelial dysfunction and vascular inflammation. A better understanding of the molecular mechanism responsible for ROS production may provide a novel target to reduce cardiovascular health burden in this setting

Adverse Epigenetic signatures by histone methyltransferase set7 contribute to vascular dysfunction in patients with type 2 diabetes

BACKGROUND: -Cellular studies showed that histone methyltransferase Set7 mediates high glucose-induced inflammation via epigenetic regulation of the transcription factor NF-kB. However, the link between Set7 and vascular dysfunction in patients with diabetes remains unknown. This study was designed to investigate whether Set7 contributes to vascular dysfunction in patients with type 2 diabetes (T2DM). METHODS AND RESULTS: -Set7-driven epigenetic changes on NF-kB p65 promoter and expression of NF-kB-dependent genes COX-2 and iNOS were assessed in peripheral blood monocytes (PBM) isolated from 68 subjects (44 T2DM patients and 24 age-matched controls). Brachial artery flow-mediated dilation (FMD), 24-hour urinary levels of 8-isoprostaglandin F2α (8-isoPGF2α) and plasma adhesion molecules ICAM-1 and MCP-1 were also determined. Experiments in human aortic endothelial cells (HAECs) exposed to high glucose were performed to elucidate the mechanisms of Set7-driven inflammation and oxidative stress. Set7 expression increased in PBM from T2DM as compared with controls. T2DM patients showed Set7-dependent monomethylation of lysine 4 of histone 3 (H3K4m1) on NF-kB p65 promoter. This epigenetic signature was associated with upregulation of NF-kB, subsequent transcription of oxidant/inflammatory genes and increased plasma levels of ICAM-1 and MCP-1. Interestingly, we found that Set7 expression significantly correlated with oxidative marker 8-isoPGF2α(r=0.38, p=0.01) and FMD (r= -0.34, p=0.04). In HAECs, silencing of Set7 prevented H3K4m1 and abolished NF-kB-dependent oxidant and inflammatory signalling. CONCLUSIONS: -Set7-induced epigenetic changes contribute to vascular dysfunction in patients with T2DM. Targeting this chromatin modifying enzyme may represent a novel therapeutic approach to prevent atherosclerotic vascular disease in this setting

Impact of glycemic variability on chromatin remodeling, oxidative stress, and endothelial dysfunction in patients with type 2 diabetes and with target HbAlevels

Intensive glycemic control (IGC) targeting HbAfails to show an unequivocal reduction of macrovascular complications in type 2 diabetes (T2D); however, the underlying mechanisms remain elusive. Epigenetic changes are emerging as important mediators of cardiovascular damage and may play a role in this setting. This study investigated whether epigenetic regulation of the adaptor protein p66, a key driver of mitochondrial oxidative stress, contributes to persistent vascular dysfunction in patients with T2D despite IGC. Thirty-nine patients with uncontrolled T2D (HbA>7.5%) and 24 age- and sex-matched healthy control subjects were consecutively enrolled. IGC was implemented for 6 months in patients with T2D to achieve a target HbAof ≤7.0%. Brachial artery flow-mediated dilation (FMD), urinary 8-isoprostaglandin F(8-isoPGF), and epigenetic regulation of p66were assessed at baseline and follow-up. Continuous glucose monitoring was performed to determine the mean amplitude of glycemic excursion (MAGE) and postprandial incremental area under the curve (AUCpp). At baseline, patients with T2D showed impaired FMD, increased urinary 8-isoPGF, and p66upregulation in circulating monocytes compared with control subjects. FMD, 8-isoPGF, and p66expression were not affected by IGC. DNA hypomethylation and histone 3 acetylation were found on the p66promoter of patients with T2D, and IGC did not change such adverse epigenetic remodeling. Persistent downregulation of methyltransferase DNMT3b and deacetylase SIRT1 may explain the observed p66-related epigenetic changes. MAGE and AUCpp but not HbAwere independently associated with the altered epigenetic profile on the p66promoter. Hence, glucose fluctuations contribute to chromatin remodeling and may explain persistent vascular dysfunction in patients with T2D with target HbAlevels

Interplay among H3K9-editing enzymes SUV39H1, JMJD2C and SRC-1 drives p66Shc transcription and vascular oxidative stress in obesity

Accumulation of reactive oxygen species (ROS) promotes vascular disease in obesity, but the underlying molecular mechanisms remain poorly understood. The adaptor p66Shc is emerging as a key molecule responsible for ROS generation and vascular damage. This study investigates whether epigenetic regulation of p66Shc contributes to obesity-related vascular disease

Targeting prolyl-isomerase Pin1 prevents mitochondrial oxidative stress and vascular dysfunction: insights in patients with diabetes

Diabetes is a major driver of cardiovascular disease, but the underlying mechanisms remain elusive. Prolyl-isomerase Pin1 recognizes specific peptide bonds and modulates function of proteins altering cellular homoeostasis. The present study investigates Pin1 role in diabetes-induced vascular disease