Phosphorylation Of Synthase Research Articles

Vascular endothelial growth factor regulation of endothelial nitric oxide synthase phosphorylation is involved in isoflurane cardiac preconditioning.

Previous studies indicate that nitric oxide derived from endothelial nitric oxide synthase (eNOS) serves as both trigger and mediator in anaesthetic cardiac preconditioning. The mechanisms underlying regulation of eNOS by volatile anaesthetics have not been fully understood. Therefore, this study examined the role of vascular endothelial growth factor (VEGF) in isoflurane cardiac preconditioning. Wistar rats underwent 30 min of coronary artery occlusion followed by 2 h of reperfusion. Isoflurane given prior to ischaemia/reperfusion significantly decreased myocardial infarct size from 60 ± 1% in control to 40 ± 3% (n = 8 rats/group, P < 0.05). The beneficial effects of isoflurane were blocked by neutralizing antibody against VEGF (nVEGF). Coronary arterial endothelial cells (ECs) alone or together with cardiomyocytes (CMs) were subjected to hypoxia/reoxygenation injury. The expression of VEGF and eNOS was analysed by western blot, and nitric oxide was measured by ozone-based chemiluminescence. In co-cultured CMs and ECs, isoflurane administered before hypoxia/reoxygenation attenuated lactate dehydrogenase activity and increased the ratio of phosphorylated eNOS/eNOS and nitric oxide production. The protective effect of isoflurane on CMs was compromised by nVEGF and after VEGF in ECs was inhibited with hypoxia inducible factor-1α short hairpin RNA (shRNA). The negative effect of hypoxia inducible factor-1α shRNA was restored by recombinant VEGF. Isoflurane cardiac preconditioning is associated with VEGF regulation of phosphorylation of eNOS and nitric oxide production.

Alterations in β‑catenin/E‑cadherin complex formation during the mechanotransduction of Saos‑2 osteoblastic cells.

Mechanical load application promotes bone formation, while reduced load leads to bone loss. However, the underlying mechanisms that regulate new bone formation are not fully understood. Wnt/β-catenin signaling has an important role in bone formation, bone growth and remodeling. The aim of the present study was to investigate whether mechanical stimuli regulated bone formation through the Wnt/β-catenin signaling pathway. Saos-2 osteoblastic cells were subjected to mechanical strain using a Flexcell strain loading system. The results demonstrated that 12% cyclical tensile stress significantly stimulated Saos-2 cell proliferation, increased the activity of alkaline phosphatase and promoted the formation of mineralized nodules, as determined by MTT and p-nitrophenyl phosphate assays and Alizarin Red S staining, respectively. Furthermore, western blot analysis demonstrated that, following mechanical strain, increased phosphorylation of glycogen synthase kinase-3β and nuclear β-catenin expression was observed in cells, compared with static control culture cells. Results of reporter gene and reverse transcription-polymerase chain reaction assays also demonstrated that mechanical strain significantly increased T-cell factor reporter gene activity and the mRNA expression of cyclooxygenase (COX)-2, cyclin D1, c-fos and c-Jun in Saos-2 cells. Co-immunoprecipitation analysis revealed that elongation mechanical strain activated Wnt/β-catenin signaling and reduced β-catenin and E-cadherin interaction in Saos-2 cells. In conclusion, the results of the current study indicate that mechanical strain may have an important role in the proliferation and differentiation of osteoblasts. The disassociation of the β-catenin/E-cadherin complex in the osteoblast membrane under stretch loading and the subsequent translocation of β-catenin into the nucleus may be an intrinsic mechanical signal transduction mechanism.

Sulfur dioxide improves endothelial dysfunction by downregulating the angiotensin II/AT1R pathway in D-galactose-induced aging rats.

The aim of this study was to investigate the protective effects of sulfur dioxide (SO2) on the endothelial function of the aorta in D-galactose (D-gal)-induced aging rats. Sprague Dawley rats were randomized into a D-gal group, a D-gal + SO2 group and a control group, then injected with D-gal, D-gal + SO2 donor or equivalent volumes of saline, respectively, for 8 consecutive weeks. After 8 weeks, the mean arterial pressure was significantly increased in the D-gal group, but was lowered by SO2. SO2 significantly ameliorated the endothelial dysfunction induced by D-gal treatment. The vasorelaxant effect of SO2 was associated with the elevated nitric oxide levels and upregulated phosphorylation of endothelial nitric oxide synthase. In the D-gal group, the concentration of angiotensin II in the plasma was significantly increased, but was decreased by SO2. Moreover, levels of vascular tissue hydrogen peroxide (H2O2) and malondialdehyde were significantly lower in SO2-treated groups than those in the D-gal group. Western blot analysis showed that the expressions of oxidative stress-related proteins (the angiotensin II type 1 receptor (AT1R), and nicotinamide adenine dinucleotide phosphate oxidase subunits) were increased in the D-gal group, while they were decreased after treatment with SO2. In conclusion, SO2 attenuated endothelial dysfunction in association with the inhibition of oxidative stress injury and the downregulation of the angiotensin II/AT1R pathway in D-gal-induced aging rats.

Ticagrelor protects against AngII-induced endothelial dysfunction by alleviating endoplasmic reticulum stress

Ticagrelor has been reported to decrease cardiovascular mortality compared with clopidogrel. This benefit cannot be fully explained by the more efficient platelet inhibition. Many studies demonstrated that ticagrelor improved endothelial function, leaving the mechanism elusive though. The present study aims to investigate whether ticagrelor protects against endothelial dysfunction induced by angiotensinII (AngII) through alleviating endoplasmic reticulum (ER) stress. Male Sprague Dawley rats were infused with AngII or vehicle and administrated with ticagrelor or vehicle for 14 days. Reactive oxygen species (ROS) was detected. Aortas from normal mice were incubated with endoplasmic reticulum stress inducer tunicamycin with or without ticagrelor. Vasorecactivity was measured on wire myography. Rat aortic endothelial cells (RAECs) were pretreated with ticagrelor followed by AngII or tunicamycin. Endothelial nitric oxide synthase (eNOS) phosphorylation and ER stress markers were determined by western blotting. Impaired endothelial function, induction of ER stress, reduced eNOS phosphorylation and elevated ROS generation was restored by ticagrelor treatment in vivo. In addition, tunicamycin induced endothelial dysfunction was improved by ticagrelor. In vitro, the induction of ER stress and inhibited eNOS phosphorylation in REACs exposed to AngII as well as tunicamycin was reversed by co-culturing with ticagrelor. In conclusion, ticagrelor protects against AngII-induced endothelial dysfunction via alleviating ER stress.

Low molecular-weight fucoidan protects against hindlimb ischemic injury in type 2 diabetic mice through enhancing endothelial nitric oxide synthase phosphorylation.

Diabetes mellitus (DM) complications are associated with ischemic injury. Angiogenesis is a therapeutic strategy for diabetic foot. The aim of this study was to investigate the possible angiogenic effect of low molecular weight fucoidan (LMWF) in diabetic peripheral arterial disease (PAD). Diabetic db/db mice and age-matched C57BL/6 mice underwent femoral artery ligation followed by LMWF (30, 60, 80 mg/kg per day, p.o.) or cilostazol (30 mg/kg/day, p.o.) treatment for 6 weeks. Endothelium-dependent vasodilation and blood flow of the hindlimb were measured. Histological and western blot analyses of CD34, vascular endothelial growth factor (VEGF), eNOS, and inflammatory factors in the gastrocnemius were performed. The effects of LMWF were confirmed in human umbilical vein endothelial cells (HUVEC). Diabetic mice with ligation exhibited hindlimb ulceration, hydrosarca, and necrosis, increased expression of inflammatory factors, and decreased levels of VEGF and eNOS phosphorylation. Treatment with LMWF markedly ameliorated foot lesions, suppressed expression of inflammatory factors, and improved plantar perfusion by promoting endothelium-dependent vasodilation and revascularization in diabetic PAD mice. In high-glucose treated HUVEC, LMWF (40 μg/mL) reversed blunted endothelial cell proliferation, migration, and tube formation, and promoted eNOS phosphorylation and VEGF expression, whereas HUVEC pretreatment with 100 μmol/L NG -nitro-l-arginine methyl ester, an eNOS antagonist, markedly inhibited the effects of LMWF. This study demonstrates that LMWF alleviates hindlimb ischemic damage, at least in part by promoting eNOS phosphorylation, nitric oxide production, and VEGF expression, resulting in enhanced angiogenesis in the ischemic region.

Macrophage Depletion Lowered Blood Pressure and Attenuated Hypertensive Renal Injury and Fibrosis.

Monocyte/macrophage recruitment is closely associated with the degree of hypertensive renal injury. We investigated the direct role of macrophages using liposome-encapsulated clodronate (LEC) to deplete monocytes/macrophages in hypertensive renal injury. C57BL/6 mice were treated with a pressor dose of angiotensin (Ang, 1.4 mg/kg/day) II plus LEC or the PBS-liposome for 2 weeks. Ang II mice developed hypertension, albuminuria, glomerulosclerosis, and renal fibrosis. LEC treatment reduced systolic blood pressure (SBP), albuminuria, and protected against renal structural injury in Ang II mice. Ang II significantly increased renal macrophage infiltration (MOMA2+ cells) and the expression of renal tumor necrosis factor α and interleukin β1, which were significantly reduced in Ang II/LEC mice. Ang II increased renal oxidative stress and the expression of profibrotic factors transforming growth factor (TGF) β1 and fibronectin. Ang II also inhibited the phosphorylation of endothelial nitric oxide synthase [phospho-endothelial nitric oxide synthesis (eNOS), ser1177]. LEC treatment reduced renal oxidative stress and TGFβ1 and fibronectin expressions, and increased phospho-eNOS expression in the Ang II mice. In Dahl rats of salt-sensitive hypertension, LEC treatment for 4 weeks significantly attenuated the elevation of SBP induced by high salt intake and protected against renal injury and fibrosis. Our results demonstrate that renal macrophages play a critical role in the development of hypertension and hypertensive renal injury and fibrosis; the underlying mechanisms may be involved in the reduction in macrophage-driven renal inflammation and restoration of the balance between renal oxidative stress and eNOS. Therefore, macrophages should be considered as a potential therapeutic target to reduce the adverse consequences of hypertensive renal diseases.

Vascular brain-derived neurotrophic factor pathway in rats with adjuvant-induced arthritis: Effect of anti-rheumatic drugs

Background and aimsIn rheumatoid arthritis, the control of both disease activity and standard cardiovascular (CV) risk factors is expected to attenuate the increased CV risk. Evidence that brain-derived neurotrophic factor (BDNF) plays a role in vascular biology led us to investigate the vascular BDNF pathway in arthritis rats as well as the interaction between endothelial nitric oxide (NO) and BDNF production. MethodsThe aortic BDNF pathway was studied in rats with adjuvant-induced arthritis, (AIA) using Western blot and immunohistochemical analysis. Control of arthritis score was achieved by administration (for 3 weeks) of an equipotent dosage of etanercept, prednisolone, methotrexate, celecoxib or diclofenac. Aortas were exposed to an NO donor or an NO synthase inhibitor and vasoreactivity experiments were performed using LM22A-4 as a TrkB agonist. ResultsVascular BDNF and full length tropomyosin-related kinase B receptor (TrkB-FL) were higher in AIA than in control rats. These changes coincided with decreased endothelial immunoreactivity in BDNF and pTrkBtyr816 and were disconnected from arthritis score. Among anti-rheumatic drugs, only prednisolone and methotrexate prevented AIA-induced vascular BDNF loss. The effect of AIA on aortic BDNF levels was reversed by an NO donor and reproduced by an NOS inhibitor. Finally, LM22A-4 induced both NO-dependent vasodilation and phosphorylation of endothelial NO synthase at serine 1177. ConclusionsOur study identified changes in the BDNF/TrkB pathway as a disease activity-independent component of AIA-associated changes in endothelial phenotype. It provides new perspectives in the understanding and management of the high CV risk reported in rheumatoid arthritis.

The Effects of Morinda citrifolia (Noni) Fruit Juice on the Prevention of Stroke by Promoting Production of Nitric Oxide through the Brain of the Spontaneously Hypertensive Stroke Prone (SHRSP) Rats

Morinda citrifolia (Noni) is a traditional folk medicinal plant and has a long history of use as a food and medicine. In order to reveal the effects of Noni fruit juice (NFJ) on stroke prevention, we performed experiments using spontaneously hypertensive stroke prone (SHRSP) rats. NFJ did not change rat body weight, food intake, and water intake. However, both systolic blood pressure (SBP) and diastolic blood pressure (DBP) were significantly decreased after NFJ treatment in SHRSP rats. Furthermore, NFJ significantly increased the survival rate, urinary nitric oxide (NO) concentration was significantly higher in the NFJ group, and endothelial NO synthase (eNOS) phosphorylation levels increased in the brain after NFJ treatment. Two pathways regulate eNOS phosphorylation: the insulin-dependent pathway and the insulin-independent pathway. For the insulin-dependent pathway, phosphorylation of insulin receptor substrate 1 (IRS1) and protein kinase B (Akt) did not change in the NFJ group. For the insulin-independent pathway, expression of adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, liver kinase B 1 (LKB1), and silent information regulator 1 (Sirt1) significantly increased in the brain of SHRSP rats after NFJ treatment. These data suggested that NFJ prevented stroke by improved blood circulation, increased NO production, and elevated eNOS phosphorylation by stimulating the insulin-independent pathway (Sirt1-LKB1-AMPK-eNOS).

Effect of SFRP5 (Secreted Frizzled–Related Protein 5) on the WNT5A (Wingless-Type Family Member 5A)-Induced Endothelial Dysfunction and Its Relevance With Arterial Stiffness in Human Subjects

SFRP5 (secreted frizzled-related protein 5) is an endogenous inhibitor of WNT5A (wingless-type family member 5a), which has been implicated in atherosclerosis. However, contradictory results have been reported about the role of SFRP5 in atherosclerosis. We aimed to investigate whether SFRP5 could restore WNT5A-induced endothelial dysfunction in vitro and ex vivo. In addition, we sought to determine whether the serum concentration of SFRP5 is associated with atherosclerosis in humans. We measured endothelium-dependent vasorelaxation in the isolated thoracic aorta of Sprague-Dawley rats. In addition, we measured intracellular nitric oxide (NO) in human endothelial cells. The protein abundance of total and phosphorylated JNK (c-Jun N-terminal kinase), AKT (protein kinase B), and endothelial NO synthase was analyzed in human endothelial cells. Circulating SFRP5 and WNT5A levels and brachial-ankle pulse wave velocity were measured in 282 human subjects with type 2 diabetes mellitus. SFRP5 dose dependently restored Wnt5-induced impaired vasorelaxation in rat thoracic aorta by an endothelial NO synthase-dependent mechanism. SFRP5 treatment restored the WNT5A-induced reduction of NO production via endothelial NO synthase in human endothelial cells. WNT5A-induced changes in the phosphorylation of JNK, AKT, and endothelial NO synthase were ameliorated with SFRP5 administration. In humans with type 2 diabetes mellitus, the serum SFRP5 concentration positively correlated with brachial-ankle pulse wave velocity (r=0.146; P=0.024). Multivariate linear regression analysis demonstrated that the serum SFRP5 concentration was independently associated with brachial-ankle pulse wave velocity after adjustment for potential confounders [B (SE)=7.40 (3.35); P=0.028]. Our data suggest the possible compensatory action of SFRP5 against atherosclerosis under conditions of metabolic dysfunction.

Sphingosine‐1‐Phosphate Type 1 Receptor and eNOS Signaling Pathway Play a Role in High Blood Pressure of Intrauterine Growth Restricted Mouse

Intrauterine growth restriction (IUGR) programs hypertension and cardiovascular (CV) disease in later life, but the underlying mechanisms remain unclear. The bioactive sphingolipid metabolite sphingosine‐1‐phosphate (S1P) is critically involved in CV development in the fetus and plays a significant role in the regulation of CV health in adulthood. S1P receptors (S1PRs) are widely expressed in CV system. We previously reported that IUGR offspring have an increase in blood pressure (BP) and have an alteration in renal S1PRs expression during‐ and post‐nephrogenesis. We also found that a nonspecific S1PRs agonist FTY720 (Fingolimod) administration decreased BP of IUGR offspring. However, which S1PR subtype and downstream signaling play a role in the decreasing of BP in IUGR is unknown. In this study, we tested the hypothesis that vascular S1P receptors type 1 (S1PR1) contributes to high BP in male IUGR mouse. Pregnant C57bl/6J mice underwent sham or reduced uterine perfusion (RUP) at day 13 of gestation with delivery at full term. IUGR offspring (from RUP dam) had a lower birth weight than control (p&lt;0.05). At 24 weeks of age, male control and IUGR mice were subjected to telemetry probes implantation, and BP was measured after one week of recovery. IUGR offspring had a significantly higher BP compared to male control, and acute administration of FTY720 (1 mg/kgBW i.p), a nonspecific S1P receptor agonist, significantly decreased BP in IUGR similar to our previously reported data. SEW2871 (3 mg/kgBW i.p), a specific S1PR1 agonist, transiently decreased BP in IUGR (7.1±2.3 mmHg, p&lt; 0.05, N=6) after one hour of treatment and the BP returned to normal level within 4 hrs. To determine the signaling pathway underlying this decreased BP after S1PR1 activation, another set of animals with SEW2871 or vehicle i.p. injections were euthanized after one hour of treatment, and the thoracic aorta was collected for western blot analysis (N=3 per group). Endothelial nitric oxide synthase (eNOS) phosphorylation at serine 1177 (p‐eNOS) and serine 473 phosphorylated Akt (p‐Akt) expressions were significantly increased in IUGR+SEW2871 vs IUGR+vehicle, p&lt; 0.05 (p‐eNOS: 2.0 fold and ; p‐Akt:1.5 fold). p‐eNOS, p‐Akt, total eNOS and total Akt were not significantly different among control+vehicle, control+SEW2871, and IUGR+vehicle groups. Phosphorylated p38 MAPK was 3 folds lower in IUGR+vehicle as compared to control+vehicle, p&lt; 0.05. SEW2871 did not up‐regulate p38 MAPK in control but significantly increased in IUGR (1.7 fold vs IUGR+vehicle, p&lt; 0.05). Phosphorylated p44/42 MAPK was not different between groups. Together our data suggest that activation of S1PR1 decrease BP in IUGR through eNOS, Akt and MAPK pathways. Thus, S1P signaling is a putative mechanism underlying the developmental programming of hypertension in IUGR mice.Support or Funding InformationDr. Intapad was supported by funding from the AHA 16SDG27770041, ASN Kidney Research Career Development grant, and Start‐up funds from Tulane University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Uric Acid Impairs Insulin Signaling by Promoting Enpp1 Binding to Insulin Receptor in Human Umbilical Vein Endothelial Cells.

High levels of uric acid (UA) are associated with type-2 diabetes and cardiovascular disease. Recent pieces of evidence attributed to UA a causative role in the appearance of diabetes and vascular damage. However, the molecular mechanisms by which UA induces these alterations have not been completely elucidated so far. Among the mechanisms underlying insulin resistance, it was reported the role of a transmembrane glycoprotein, named either ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) or plasma cell antigen 1, which is able to inhibit the function of insulin receptor (IR) and it is overexpressed in insulin-resistant subjects. In keeping with this, we stimulated human umbilical vein endothelial cells (HUVECs) with insulin and UA to investigate the effects of UA on insulin signaling pathway, testing the hypothesis that UA can interfere with insulin signaling by the activation of ENPP1. Cultures of HUVECs were stimulated with insulin, UA and the urate transporter SLC22A12 (URAT1) inhibitor probenecid. Akt and endothelial nitric oxide synthase (eNOS) phosphorylation levels were investigated by immunoblotting. ENPP1 binding to IR and its tyrosine phosphorylation levels were tested by immunoprecipitation and immunoblotting. UA inhibited insulin-induced Akt/eNOS axis. Moreover, UA induced ENPP1 binding to IR that resulted in an impairment of insulin signaling cascade. Probenecid reverted UA effects, suggesting that UA intracellular uptake is required for its action. In endothelial cells, UA directly interferes with insulin signaling pathway at receptor level, through ENPP1 recruitment. This evidence suggests a new molecular model of UA-induced insulin resistance and vascular damage.

Inhibition of endothelial nitric oxide synthase reverses the effect of exercise on improving cognitive function in hypertensive rats.

Hypertension-induced endothelial dysfunction is associated with β-amyloid (Aβ) deposition, a typical pathology of Alzheimer's disease (AD). Endothelial nitric oxide synthase (eNOS) phosphorylation, impaired by phosphatidylinositol 3-kinase (PI3K)/protein kinase-B(Akt) pathway abnormalities in hypertensive rats, has a critical role in endothelial function. However, it is unknown whether eNOS participates in the hypertension-induced pathology of AD. In this study, we investigated the role of eNOS in Aβ deposition and cognitive function in stroke-prone spontaneously hypertensive (SHRSP) rats. Physical exercise was used as a promoter, and Nω-nitro L-arginine methyl ester (L-NAME) was used as an inhibitor of eNOS to determine the effects of eNOS on SHRSP rats. Compared with Wistar Kyoto (WKY) rats, the hypertensive challenge caused cognitive impairment, decreased eNOS levels and increased amyloid precursor protein (APP), β-secretase, and Aβ levels in the cortex and hippocampus. Sixteen weeks of exercise lowered blood pressure (BP), promoted eNOS expression, ameliorated Alzheimer's pathology, and improved cognitive function in 29-week-old SHRSP rats. Furthermore, daily treatment with L-NAME reversed the beneficial effects of exercise on SHRSP rats. Exercise also decreased the protein levels of insulin-like growth factor-1 (IGF-1), PI3K, and phospho-Akt (p-Akt, ser473). In addition, long-term exercise increased the expression levels of IGF-1, PI3K, and p-Akt (ser473) in the brains of SHRSP rats. In conclusion, eNOS downregulation contributed to hypertension-induced Alzheimer pathology and cognitive impairment. Long-term exercise initiated in rats at a young age promoted eNOS expression and attenuated vascular-related Alzheimer's pathology via the IGF-1/PI3K/p-Akt pathway in SHRSP rats.

Colonic metabolites from flavanols stimulate nitric oxide production in human endothelial cells and protect against oxidative stress-induced toxicity and endothelial dysfunction

Oxidative stress is involved in endothelial dysfunction, the key player in the development of vascular events. Flavanols, the major antioxidants in cocoa have been related to vascular protection and lower cardiovascular risk. However, the bioavailability of cocoa flavanols is very low and their bioactivity in vivo seems to be greatly mediated by the derived phenolic metabolites formed by intestinal microbiota. Hence, we investigated whether microbial-derived flavanol metabolites 3,4-dihydroxyphenylacetic acid (DHPAA), 2,3-dihydroxybenzoic acid (DHBA), 3-hydroxyphenylpropionic acid (HPPA) and a mix of them could influence endothelial function and prevent oxidative stress in human endothelial cells (Ea.hy926). Our results revealed that a mixture of flavanol colonic metabolites significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. By using specific inhibitors, we also established the participation of the adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) in eNOS activation. Likewise, flavanol metabolite mix protected against oxidative stress-induced endothelial dysfunction and cell death by preventing increased ROS generation and activation of signaling pathways related to oxidative stress. We concluded that flavanol colonic metabolites could exert beneficial effects in endothelial cells and prevent oxidative stress-induced vascular dysfunction.

Propylthiouracil-coated biodegradable polymer inhibited neointimal formation and enhanced re-endothelialization after vascular injury.

BackgroundThe drug-eluting stent is a standard approach for the treatment of coronary artery disease. Propylthiouracil (PTU), an antithyroid drug, has been proven to suppress neointimal formation after balloon injury.Materials and methodsThis study used a biodegradable polymer coating with PTU to test its effects on platelet function, re-endothelialization, and neointimal formation after vascular injury. Electrospinning was used to fabricate hybrid stents and generate PTU-loaded nanofibers.ResultsPTU-eluting stents maintained a stable release of PTU for 3 weeks. The PTU-coated stent markedly decreased the neointimal formation induced by vascular injury in the descending aorta of rabbits. Moreover, the PTU coating reduced platelet adhesion on the surface of the biodegradable membrane, which was reflected by the decreased expression of adhesion molecule in PTU-treated endothelial cells. The PTU coating enhanced re-endothelialization in injured aortas. In vitro, PTU exerted less suppressive effect on the proliferation and migration of endothelial cells than on those of vascular smooth muscle cells. Furthermore, treatment of endothelial cells with PTU induced phosphorylation (Ser1177) of endothelial nitric oxide synthase as well as its association with heat shock protein 90, supporting the protective role of PTU in endothelial function. The level of thyroid-stimulating hormone remained unchanged during the experimental period.ConclusionThis study indicates that PTU can be released locally and steadily in injured aortas, with some local effects but without systemic effects. Furthermore, PTU-coated stents may have beneficial effects on neointimal formation, endothelial cell, and platelet functions.

Resveratrol regulates blood pressure by enhancing AMPK signaling to downregulate a Rac1-derived NADPH oxidase in the central nervous system.

Resveratrol is a polyphenol with pleiotropic effects against oxidative damage that has been widely implicated in lowering hypertension risk. The purpose of this study was to determine whether improve nitric oxide (NO) release in the brain, either through the activation of AMP-activated protein kinase (AMPK) or reduced Ras-related C3 botulinum toxin substrate 1 (Rac1)-induced reactive oxygen species (ROS) generation, thereby reducing blood pressure (BP) in rats with fructose-induced hypertension. The rats were fed with 10% fructose or Crestor (rosuvastatin; 1.5 mg·kg-1·day-1) and resveratrol (10 mg·kg-1·day-1) treatment for 1 wk, then the systolic blood pressure of the rats was measured by tail-cuff method. Endogenous in vivo superoxide radical production in the nucleus tractus solitarii (NTS) was determined with dihydroethidium. Immunoblotting analyses were used to quantify protein expression levels. Oral resveratrol treatment for 1 wk decreased BP and increased NO production in the NTS of fructose-fed rats but not in the control Wistar-Kyoto rats. The effect of Crestor is opposite that of resveratrol. Fructose induced hypertension by inactivating AMPK, which in turn enhanced the generation of ROS and reduced manganese superoxide dismutase by increasing the activity of Rac1-induced NADPH oxidase, abolishing the activity of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and ribosomal protein S6 kinase (RSK) and neuronal nitric oxide synthase (nNOS) phosphorylation signaling pathway in the brain. However, resveratrol had the opposite effect in the fructose-fed rats. Overall, we show that the resveratrol decreased BP better than Crestor, abolished ROS generation, and enhanced the ERK1/2-RSK-nNOS pathway by activating AMPK to downregulate Rac1-induced NADPH oxidase levels in the NTS during oxidative stress-associated hypertension. NEW & NOTEWORTHY 1) Evidence showed that the Ras-related C3 botulinum toxin substrate 1 (Rac1) augmented by Crestor (rosuvastatin) did not result in a significant change in blood pressure (BP) in fructose-induced hypertension. 2) Fructose induced hypertension by inactivating AMP-activated protein kinase (AMPK), which in turn enhanced the generation of reactive oxygen species (ROS) and reduced manganese superoxide dismutase in the brain. 3) Resveratrol decreased BP better than Crestor, abolished ROS generation, and enhanced the ERK1/2-ribosomal protein S6 kinase-neuronal nitric oxide synthase pathway by activating AMPK to negatively regulate Rac1-induced NADPH oxidase levels in the nucleus tractus solitarii during oxidative stress-associated hypertension.

Cold acclimation reduces hepatic protein Kinase B and AMP-activated protein kinase phosphorylation and increases gluconeogenesis in Rats.

This study investigated the molecular and metabolic responses of the liver to cold‐induced thermogenesis. To accomplish that, male Wistar rats were exposed to cold (4°C) for 7 days. Livers were then extracted and used for the determination of glucose and fatty acid oxidation, glycogen content, the expression and content of proteins involved in insulin signaling, as well as in the regulation of gluconeogenesis and de novo lipid synthesis. Despite being hyperphagic, cold‐acclimated rats displayed normoglycemia with reduced insulinemia, which suggests improved whole‐body insulin sensitivity. However, liver protein kinase B (AKT) and glycogen synthase kinase 3 (GSK3) phosphorylations were markedly reduced along with the expressions of the insulin receptor (IR) and its substrates IRS1 and IRS2, whereas glycogen synthase (GS) phosphorylation increased. Thus, major signaling steps of the glycogen synthesis pathway in the liver were inhibited. Furthermore, glucagonemia and hepatic glucose and fatty acid oxidation were increased, whereas liver glycogen content was reduced by cold acclimation. This was accompanied by significantly elevated expressions of the gluconeogenic transcription regulators CRTC2, PGC‐1α, and FoxO1, as well as of major gluconeogenic enzymes (G6Pase, FBP1, and PEPCK). Conversely, phosphorylation and contents of AMP‐activated protein kinase (AMPK) and acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS) content were markedly downregulated in livers of cold‐acclimated rats. In conclusion, cold acclimation suppressed hepatic glycogen synthesis and promoted profound metabolic changes in the liver so the organ could sustain its ability to regulate whole‐body glucose and lipid metabolism under conditions of high‐energy demand in thermogenic tissues.

Cardioprotection by Metformin: Beneficial Effects Beyond Glucose Reduction.

Metformin is a biguanide that is widely used as an insulin-sparing agent to treat diabetes. When compared with the general population, diabetics are twice as likely to die from fatal myocardial infarction and congestive heart failure (CHF). There has been a significant concern regarding the use of metformin in patients with CHF because of their higher tendency to develop lactic acidosis. However, large epidemiological trials have reported better cardiovascular prognosis with metformin compared to other glucose-lowering agents among diabetics. Additionally, metformin has reduced the risk of reinfarction and all-cause mortality in patients with coronary artery disease and CHF, respectively. The protection against cardiovascular diseases appears to be independent of the anti-hyperglycemic effects of metformin. These effects are mediated through an increase in 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation and by increased phosphorylation of endothelial nitric oxide synthase (eNOS) in cardiomyocytes with an increased production of nitric oxide (NO). Metformin preconditions the heart against ischemia-reperfusion injury and may improve myocardial remodeling after an ischemic insult. The preponderance of evidence currently suggests that metformin is safe in patients with CHF, prompting the Food and Drug Administration to remove CHF as a contraindication from the package insert of all generic metformin preparations. In this narrative, along with a limited meta-analysis of available studies, we have reviewed the pleiotropic (non-glucose-lowering) effects of metformin that potentially contribute to its cardioprotective properties. Additionally, we have reviewed issues surrounding the safety of metformin in patients with cardiac diseases.

Cinacalcet-mediated activation of the CaMKK\u03b2-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

Apoptosis and autophagy are harmoniously regulated biological processes for maintaining tissue homeostasis. AMP-activated protein kinase (AMPK) functions as a metabolic sensor to coordinate cellular survival and function in various organs, including the kidney. We investigated the renoprotective effects of cinacalcet in high-glucose treated human glomerular endothelial cells (HGECs), murine podocytes and C57BLKS/J-db/db mice. In cultured HGECs and podocytes, cinacalcet decreased oxidative stress and apoptosis and increased autophagy that were attributed to the increment of intracellular Ca2+ concentration and the phosphorylation of Ca2+/calmodulin-dependent protein kinase kinaseβ (CaMKKβ)-Liver kinase B1 (LKB1)-AMPK and their downstream signals including the phosphorylation of endothelial nitric oxide synthase (eNOS) and increases in superoxide dismutases and B cell leukemia/lymphoma 2/BCL-2-associated X protein expression. Interestingly, intracellular chelator BAPTA-AM reversed cinacalcet-induced CaMKKβ elevation and LKB1 phosphorylation. Cinacalcet reduced albuminuria without influencing either blood glucose or Ca2+ concentration and ameliorated diabetes-induced renal damage, which were related to the increased expression of calcium-sensing receptor and the phosphorylation of CaMKKβ-LKB1. Subsequent activation of AMPK was followed by the activation of peroxisome proliferator-activated receptor γ coactivator-1α and phospho-Ser1177eNOS-nitric oxide, resulting in a decrease in apoptosis and oxidative stress as well as an increase in autophagy.Our results suggest that cinacalcet increases intracellular Ca2+ followed by an activation of CaMKKβ-LKB1-AMPK signaling in GECs and podocytes in the kidney, which provides a novel therapeutic means for type 2 diabetic nephropathy by modulation of apoptosis and autophagy.

Emodin, A Chinese Herbal Medicine, Inhibits Reoxygenation-Induced Injury in Cultured Human Aortic Endothelial Cells by Regulating the Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathway.

BackgroundIschemia-reperfusion injury is associated with vascular dysfunction. The aim of this study was to investigate the role of emodin, a Chinese herbal medicine, in hypoxia-reoxygenation injury in cultured human aortic endothelial cells (HAECs) and its effects on the expression of the peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS) signaling pathway.Material/MethodsAn in vitro hypoxia-reoxygenation model used cultured human aortic endothelial cells (HAECs). A colorimetric method evaluated the activity of peroxisome proliferator-activated receptor-γ (PPAR-γ). Phosphorylation of PPAR-γ and endothelial nitric oxide synthase (eNOS) were measured by Western blotting. Expression of inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Nitric oxide (NO) production was detected by diaminofluorescein-FM diacetate (DAF-FM DA) fluorescence. Immunoprecipitation was used to evaluate the molecular coupling of heat shock protein (HSP)90 and eNOS.ResultsHypoxia-reoxygenation injury of HAECs reduced the activity and phosphorylation of PPAR-γ, and eNOS, NO production, and HSP90/eNOS molecular coupling in a time-dependent manner. Hypoxia-reoxygenation increased the levels of inflammatory cytokines TNF-α, IL-6, and IL-8 in a time-dependent manner. Emodin treatment recovered PPAR-γ activity and phosphorylation, eNOS phosphorylation, and HSP90/eNOS coupling in HAECS in a concentration-dependent manner, which was reversed by the PPAR-γ inhibitor GW9662, and the eNOS inhibitor, L-NAME. The recovery of HSP90/eNOS coupling by emodin was impaired by GW9662 treatment.ConclusionsAn in vitro hypoxia-reoxygenation (ischemia-reperfusion injury) model of induction of endothelial cell inflammatory mediators showed that emodin recovered the PPAR-γ and eNOS pathway activity.

Dexmedetomidine Maintains Its Direct Cardioprotective Effect Against Ischemia/Reperfusion Injury in Hypertensive Hypertrophied Myocardium.

Dexmedetomidine (DEX) has a direct cardioprotective effect against ischemia/reperfusion injury through endothelial nitric oxide synthase (eNOS) phosphorylation via α2-adrenoreceptor (α2-AR). By using spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat models, the cardioprotective effect of DEX in hypertrophied myocardium and the differential characteristics of cardiac α2-AR and the I1 imidazoline receptor (I1R) were examined. Langendorff-perfused rat hearts underwent 40 minutes of global ischemia followed by 120 minutes of reperfusion in the presence or absence of DEX before ischemia. Infarct size was measured, and eNOS phosphorylation was assessed by Western blotting. The presence and expression of the receptors were assessed by immunohistochemistry, real-time reverse transcriptase polymerase chain reaction, and Western blotting. In WKY, DEX significantly decreased infarct size and increased phosphorylated-eNOS/eNOS. These effects were counteracted by yohimbine (α2-AR antagonist) and efaroxan (α2-AR and I1R antagonist). In SHR, DEX significantly decreased infarct size, and the effect was counteracted by efaroxan but not yohimbine. DEX did not alter phosphorylated-eNOS/eNOS in SHR. α2-AR and I1R were observed in WKY and SHR hearts. Although alpha2A-AR and alpha2B-AR messenger RNA and protein levels were upregulated in SHR, I1R expression was comparable between the 2 species. In the hypertrophied heart, DEX maintains its direct cardioprotective effect against ischemia/reperfusion injury via I1R in an eNOS-nondependent manner despite upregulation of α2-AR.