Thrombin Signaling Contributes to High Glucose-Induced Injury of Human Brain Microvascular Endothelial Cells.

BACKGROUND: Diabetes is one of the strongest disease-related risk factors for Alzheimer's disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. OBJECTIVE: Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. METHODS: Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30 mM glucose±100 nM thrombin and±250 nM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. RESULTS: reatment of HBMVECs with 30 mM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. CONCLUSION: Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteins and a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

Sevoflurane Effect on Cognitive Function and the Expression of Oxidative Stress Response Proteins in Elderly Patients undergoing Radical Surgery for Lung Cancer.

OBJECTIVE: To investigate the effects of sustained inhalation of sevoflurane on cognitive function and the expression of oxidative stress response proteins such as NADPH oxidase subunits NOX2 and NOX4 in elderly patients undergoing radical surgery for lung cancer. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Department of Anesthesiology, Suzhou Kowloon Hospital, Shanghai Jiao Tong University School of Medicine, China, from February 2016 to October 2017. METHODOLOGY: Elderly patients who underwent radical surgery for lung cancer were divided into the sevoflurane group and the propofol group, with 52 cases in each group. Sustained inhalation of sevoflurane and propofol was administered to maintain anesthesia in the respective groups. Cognitive function and lung function parameters were compared between the two groups. Serum S100 β levels and expression of NOX2 and NOX4 proteins in peripheral blood mononuclear cells of the two groups were determined. RESULTS: At 24 hours after surgery, the lung function indices of the sevoflurane group such as FEV1, FVC and VC were higher than those of the propofol group (p<0.001, p=0.008 and p=0.002, respectively). At the end of the surgery and at 24 hours after surgery, the MMSE scores of the sevoflurane group were higher than the propofol group (all p<0.001). S100 levels were lower than the propofol group (p=0.003 and p<0.001, respectively). Levels of NADPH oxidase subunits NOX2 and NOX4 proteins in peripheral blood mononuclear cells of the sevoflurane group were lower than the propofol group (p=0.033, p<0.001, p<0.001and p<0.001, respectively). CONCLUSION: Compared with intravenous anesthesia with propofol, general anesthesia with sevoflurane inhalation has little effect on the short-term cognitive function in elderly patients undergoing radical surgery for lung cancer, and can effectively improve lung function. The mechanism may be related to the reduction of the expression of NOX2 and NOX4 proteins.

Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes.

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat-an NO-independent activator of oxidized guanylate cyclase-increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4-dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

Hydrogen Peroxide-Responsive Nanoparticle Reduces Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R), a large amount of reactive oxygen species (ROS) is produced. In particular, overproduction of hydrogen peroxide (H2O2) is considered to be a main cause of I/R-mediated tissue damage. We generated novel H2O2-responsive antioxidant polymer nanoparticles (PVAX and HPOX) that are able to target the site of ROS overproduction and attenuate the oxidative stress-associated diseases. In this study, nanoparticles were examined for their therapeutic effect on myocardial I/R injury. METHODS AND RESULTS: The therapeutic effect of nanoparticles during cardiac I/R was evaluated in mice. A single dose of PVAX (3 mg/kg) showed a significant improvement in both cardiac output and fraction shortening compared with poly(lactic-coglycolic acid) (PLGA) particle, a non-H2O2-activatable nanoparticle. PVAX also significantly reduced the myocardial infarction/area compared with PLGA (48.7±4.2 vs 14.5±2.1). In addition, PVAX effectively reduced caspase-3 activation and TUNEL-positive cells compared with PLGA. Furthermore, PVAX significantly decreased TNF-α and MCP-1 mRNA levels. To explore the antioxidant effect of PVAX by scavenging ROS, dihydroethidium staining was used as an indicator of ROS generation. PVAX effectively suppressed the generation of ROS caused by I/R, whereas a number of dihydroethidium-positive cells were observed in a group with PLGA I/R. In addition, PVAX significantly reduced the level of NADPH oxidase (NOX) 2 and 4 expression, which favors the reduction in ROS generation after I/R. CONCLUSIONS: Taken together, these results suggest that H2O2-responsive antioxidant PVAX has tremendous potential as a therapeutic agent for myocardial I/R injury.