5-MTHF enhances the portal pressure reduction achieved with propranolol in patients with cirrhosis: A randomized placebo-controlled trial.

BACKGROUND & AIMS: ß-blockers reduce hepatic venous pressure gradient (HVPG) by decreasing portal inflow, with no reduction in intrahepatic vascular resistance. 5-Methyltetrahydrofolate (5-MTHF) can prevent oxidative loss of tetrahydrobiopterin (BH4), a cofactor for endothelial nitric oxide synthase coupling. It also converts homocysteine (tHcy) into methionine and enables the degradation of asymmetric dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase. The aim of this study was to evaluate the effects of 5-MTHF in combination with propranolol on HVPG and nitric oxide bioavailability markers in patients with cirrhosis and portal hypertension. METHOD: Sixty patients with cirrhosis and HVPG ≥12 mmHg were randomized 1:1 to receive treatment with 5-MTHF+propranolol or placebo+propranolol for 90 days under double-blind conditions. HVPG and markers of nitric oxide bioavailability (BH4, ADMA and tHcy) were measured again at the end of treatment. RESULTS: Groups were similar in terms of baseline clinical and hemodynamic data and nitric oxide bioavailability markers. HVPG decreased in both groups, but the magnitude of the change was significantly greater in the group treated with 5-MTHF+propranolol compared to placebo+propranolol (percentage decrease, 20 [29-9] vs. 12.5 [22-0], p = 0.028), without differences in hepatic blood flow. At the end of treatment, 5-MTHF+propranolol (vs. placebo+propranolol) was associated with higher BH4 (1,101.4 ± 1,413.3 vs. 517.1 ± 242.8 pg/ml, p <0.001), lower ADMA (109.3 ± 52.7 vs. 139.9 ± 46.7 µmol/L, p = 0.027) and lower tHcy (µmol/L, 11.0 ± 4.6 vs. 15.4 ± 7.2 µmol/L, p = 0.010) plasma levels. CONCLUSION: In patients with cirrhosis and portal hypertension, 5-MTHF administration significantly enhanced the HVPG reduction achieved with propranolol. This effect appears to be mediated by improved nitric oxide bioavailability in the hepatic microcirculation. CLINICAL TRIAL EUDRACT NUMBER: 2014-002018-21. IMPACT AND IMPLICATIONS: Currently, the pharmacological prevention of cirrhosis complications due to portal hypertension, such as esophageal varices rupture, is based on the use of ß-blockers, but some patients still present with acute variceal bleeding, mainly due to an insufficient reduction of portal pressure. In this study, we sought to demonstrate that the addition of folic acid to ß-blockers is more effective in reducing portal pressure than ß-blockers alone. This finding could represent the basis for validation studies in larger cohorts, which could impact the future prophylactic management of variceal bleeding in cirrhosis. Enhancing the benefit of ß-blockers with a safe, accessible, cost-effective drug could improve clinical outcomes in cirrhosis, which in turn could translate into a reduction in the rates and costs of hospitalization, and ultimately into improved survival.

Combined Pulsed Magnetic Field and Radiofrequency Electromagnetic Field Enhances MMP-9, Collagen-4, VEGF Synthesis to Improve Wound Healing Via Hif-1α/eNOS Pathway.

BACKGROUND: The blood supply of the tissue is very important in the acceleration of wound healing. Radiofrequency electromagnetic field (RF) and the pulsed magnetic field (PMF) increase vasodilation to contribute wound healing. The aim of this study was to evaluate the effects of RF and PMF on wound healing via hypoxia-inducible factor-1 alpha (Hif-1α)/endothelial nitric oxide synthase (eNOS) pathway. METHODS: Forty-eight rats were divided into 4 groups as sham (wound created only), PMF (27.12 MHz, 12 times a day at 30-min intervals), RF (0.5 mT, continuously) and PMF + RF groups. Wounds were created at 1.5 × 1.5 cm size to the dorsal region, and animals were put into unit. Six animals were killed on days 4 and 7; wound tissues were collected for histopathological, immunohistochemical as collagen-4, cytokeratin, matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) staining and Hif-1α/eNOS/VEGF expressions. RESULTS: On day 4, in addition to increasing VEGF and MMP-9 stainings, connection between intact tissue and scar tissue which was stronger in the RF- and PMF-applied groups was observed. On day 7, epithelization started; inflammatory reaction decreased; collagen production, cytokeratin, VEGF and MMP-9 expression enhanced, especially in the RF + PMF applied group. eNOS, Hif-1α and VEGF expression levels were found to be significantly highest in both days of RF + PMF-applied group. CONCLUSIONS: This study revealed that both in vitro RF and PMF applications can cause notable changes in factors that are required for tissue repair on wound healing such as epithelization, connective tissue formation, collagen production and angiogenesis via vasodilatory Hif-1α/eNOS pathway and VEGF signaling. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Cholesterol efflux regulator ABCA1 exerts protective role against high shear stress-induced injury of HBMECs via regulating PI3K/Akt/eNOS signaling.

BACKGROUND: In brain, microvascular endothelial cells are exposed to various forces, including shear stress (SS). However, little is known about the effects of high shear stress (HSS) on human brain microvascular endothelial cells (HBMECs) and the underlying mechanism. The cholesterol efflux regulator ATP-binding cassette subfamily A member 1 (ABCA1) has been demonstrated to exert protective effect on HBMECs. However, whether ABCA1 is involved in the mechanism underneath the effect of HSS on HBMECs remains obscure. In the present study, a series of experiments were performed to better understand the effect of HSS on cellular processes of HBMECs and the possible involvement of ABCA1 and PI3K/Akt/eNOS in the underlying mechanisms. RESULTS: HBMECs were subjected to physiological SS (PSS) or high SS (HSS). Cell migration was evaluated using Transwell assay. Apoptotic HBMECs were detected by flow cytometry or caspase3/7 activity. IL-1ß, IL-6, MCP-1 and TNF-α levels were measured by ELISA. RT-qPCR and western blotting were used for mRNA and protein expression detection, respectively. ROS and NO levels were detected using specific detection kits. Compared to PSS, HBMECs exhibited decreased cell viability and migration and increased cell apoptosis, increased levels of inflammatory cytokines, and improved ROS and NO productions after HSS treatment. Moreover, HSS downregulated ABCA1 but upregulated the cholesterol efflux-related proteins MMP9, AQP4, and CYP46 and activated PI3K/Akt/eNOS pathway. Overexpression of ABCA1 in HBMECS inhibited PI3K/Akt/eNOS pathway and counteracted the deleterious effects of HSS. Contrary effects were observed by ABCA1 silencing. Inhibiting PI3K/Akt/eNOS pathway mimicked ABCA1 effects, suggesting that ABCA1 protects HBMECs from HSS via PI3K/Akt/eNOS signaling. CONCLUSION: These results advanced our understanding on the mechanisms of HSS on HBMECs and potentiated ABCA1/PI3K/Akt/eNOS pathway as therapeutic target for cerebrovascular diseases.

Cumulative effect of simvastatin, L-arginine, and tetrahydrobiopterin on cerebral blood flow and cognitive function in Alzheimer's disease.

BACKGROUND AND OBJECTIVES: Vascular disease is a known risk factor for Alzheimer's disease (AD). Endothelial dysfunction has been linked to reduced cerebral blood flow. Endothelial nitric oxide synthase pathway (eNOS) upregulation is known to support endothelial health. This single-center, proof-of-concept study tested whether the use of three medications known to augment the eNOS pathway activity improves cognition and cerebral blood flow (CBF). METHODS: Subjects with mild AD or mild cognitive impairment (MCI) were sequentially treated with the HMG-CoA reductase synthesis inhibitor simvastatin (weeks 0-16), L-arginine (weeks 4-16), and tetrahydrobiopterin (weeks 8-16). The primary outcome of interest was the change in CBF as measured by MRI from baseline to week 16. Secondary outcomes included standard assessments of cognition. RESULTS: A total of 11 subjects were deemed eligible and enrolled. One subject withdrew from the study after enrollment, leaving 10 subjects for data analysis. There was a significant increase in CBF from baseline to week 8 by ~13% in the limbic and ~15% in the cerebral cortex. Secondary outcomes indicated a modest but significant increase in the MMSE from baseline (24.2±3.2) to week 16 (26.0±2.7). Exploratory analysis indicated that subjects with cognitive improvement (reduction of the ADAS-cog 13) had a significant increase in their respective limbic and cortical CBF. CONCLUSIONS: Treatment of mild AD/MCI subjects with medications shown to augment the eNOS pathway was well tolerated and associated with modestly increased cerebral blood flow and cognitive improvement. TRIAL REGISTRATION: This study is registered in https://www. CLINICALTRIALS: gov ; registration identifier: NCT01439555; date of registration submitted to registry: 09/23/2011; date of first subject enrollment: 11/2011.

[Effects of 4'-O-methylochnaflavone on endothelial dysfunction induced by palmitic acid in rat cavernous endothelial cells].

OBJECTIVE: To investigate the effect of biflavonoid 4'-O-methylochnaflavone (MF) on palmitic acid-induced endothelial dysfunction in rat cavernous endothelial cells (RCECs). METHODS: The isolated RCECs were commercially available and randomly divided into four groups: normal+BSA group (NC group), palmitic acid (PA) group, MF group, and icariside Ⅱ (ICA Ⅱ) group. The protein expression levels of protein kinase B (PKB/AKT) and endothelial nitric oxide synthase (eNOS) in each group were evaluated via Western blotting. The differences in the intracellular nitric oxide of RCECs treated by MF or ICA Ⅱ were detected by DAF-FM DA that served as a nitric oxide fluorescent probe. Effects of MF and ICA Ⅱ on cell proliferation of PA-stimulated RCECs were determined via CCK-8 assay. RESULTS: The content of nitric oxide in RCECs was significantly increased after the treatment of MF and ICA Ⅱ in comparison with the NC group (P < 0.05). Moreover, compared with ICA Ⅱ group, MF demonstrated a more obvious effect in promoting nitric oxide production (P < 0.05). Compared with the NC group, the expression levels of eNOS and AKT in the PA group were significantly decreased, indicating that a model for simulating the high-fat environment in vitro was successfully constructed (P < 0.05). Meanwhile, the intervention of MF and ICA Ⅱ could effectively increase the expression of eNOS and AKT, suggesting that MF and ICA Ⅱ could promote the recovery of endothelial dysfunction caused by high levels of free fatty acids (P < 0.05). The results of CCK-8 assays showed that PA could significantly reduce the proli-feration ability of RCECs (P < 0.05). Furthermore, the decreased cell viability induced by PA was significantly elevated by treatment with ICA Ⅱ and MF (P < 0.05). CONCLUSION: In RCECs, MF and ICA Ⅱ could effectively increase the content of nitric oxide. The down-regulation of the expression of proteins associated with the AKT/eNOS pathway after PA treatment revealed that this pathway was involved in the development of endothelial dysfunction, which could be effectively reversed by MF and ICA Ⅱ. In addition, the cell proliferation ability was significantly decreased following PA treatment, but MF and ICA Ⅱ could restore the above changes. Overall, biflavonoid MF has an obvious repairing effect on PA-stimulated endothelial dysfunction.

Effects of aldo-keto reductase family 1 member A on osteoblast differentiation associated with lactate production in MC3T3-E1 preosteoblastic cells.

Aldo-keto reductase family 1 member A (AKR1A) is an NADPH-dependent aldehyde reductase widely expressed in mammalian tissues. In this study, induced differentiation of MC3T3-E1 preosteoblasts was found to increase AKR1A gene expression concomitantly increased NOx- (nitrite + nitrate), increased glucose uptake, increased [NAD(P)+]/[NAD(P)H] and lactate production but decreased reactive oxygen species (ROS) without changes in endothelial nitric oxide synthase (eNOS) expression in differentiated osteoblasts (OBs). A study using gain- and loss-of-function MC3T3-E1 cells indicated that AKR1A is essential for modulating OB differentiation and gene expression of collagen 1 A1, receptor activator of nuclear factor kappa-B ligand, and osteoprotegerin in OBs. Immunofluorescence microscopy also revealed that changes in AKR1A expression altered extracellular collagen formation in differentiated OBs. Consistently, analyses of alkaline phosphatase activity and calcium deposits of matrix mineralization by Alizarin Red S staining verified that AKR1A is involved in the regulation of OB differentiation and bone matrix formation. In addition, AKR1A gene alterations affected the levels of NOx-, eNOS expression, glucose uptake, [NAD(P)+]/[NAD(P)H] dinucleotide redox couples, lactate production, and ROS in differentiated OBs. Herein, we report that AKR1A-mediated denitrosylation may play a role in the regulation of lactate metabolism as well as redox homeostasis in cells, providing an efficient way to quickly gain energy and to significantly reduce oxidative stress for OB differentiation.

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats.

OBJECTIVE: To investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs). METHODS: Eight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta. RESULTS: EA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects. CONCLUSION: EA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.

Protective Role of ß-Sitosterol in Glucocorticoid-Induced Osteoporosis in Rats Via the RANKL/OPG Pathway.

Introduction: Osteoporosis affects approximately 10% of the population worldwide. ß-sitosterol (BSS), a major phytosterol in plants, has been claimed for centuries to have numerous medical benefits, including bone strengthening. This study aimed to find the benefit of BSS in treating osteoporosis according to traditional methods and to investigate the protective effect of BSS on glucocorticoid-induced osteoporosis in rats. Design: Wistar rats were randomly assigned to one of four groups: the control group, the dexamethasone (DEX) group and one of two BSS-treated osteoporosis groups (100 and 200 mg/kg). Blood samples and femur bones were collected for histopathology, immunohistochemistry, biochemical and mRNA expression analysis. Results: The results indicated that BSS (100 and 200 mg/kg) increased bone length, bone weight and bone mineral density (BMD) and suppressed DEX-induced reduction in body weight, dose-dependently. Mechanistically, BSS (100 and 200 mg/kg) treatment alleviated the increase of bone resorption markers and the decline of osteogenic markers, which might be partially mediated by regulation of nuclear factor kappa-ß ligand/osteoprotegerin (RANKL/OPG) and RunX2 pathways. The immunohistochemical inducible nitric oxide synthase (iNOS) results of the rats' distal femur were negative in all groups. However, except in the DEX group, the endothelial nitric oxide synthase (eNOS) color reaction in osteoblasts was strongly positive in the other 3 groups. These results suggest that BSS showed promising effects in protection against glucocorticoid-induced osteoporosis by protecting osteoblasts and suppressing osteoclastogenesis.

Formononetin regulates endothelial nitric oxide synthase to protect vascular endothelium in deep vein thrombosis rats.

OBJECTIVE: Formononetin is a bioactive isoflavone that has numerous medicinal benefits. We explored the feasibility and its mechanism of formononetin on treating acute deep vein thrombosis (DVT) in rats. MATERIALS AND METHODS: Inferior vena cava (IVC) stenosis was performed to establish the DVT rat model. First, different doses of formononetin were used to observe the feasibility of formononetin on treating DVT. In sham and DVT groups, rats were orally treated with vehicle. In the remaining groups, formononetin (10 mg/kg, 20 mg/kg, and 40 mg/kg) was orally treated once a day for 7 days at 24 h after IVC. After 7 days, the levels of thrombosis and inflammation related factors in plasma were measured. The expression of endothelial nitric oxide synthase (eNOS) was analyzed by western blot and immunofluorescence. Molecular docking was used to evaluate the interaction between the formononetin and eNOS. Further, the NOS inhibitor (L-NAME) was used to explore the mechanism of formononetin for DVT. RESULT: After treatment with formononetin, the average weights of thrombosis were decreased, and the levels of thrombosis and inflammation related factors were also significantly decreased. Additionally, phosphorylation of eNOS was increased with the formononetin administration. There is a good activity of formononetin to eNOS (total score = -6.8). However, the effects of 40 mg/kg formononetin were concealed by the NOS inhibitor (L-NAME). CONCLUSION: Formononetin reduces vascular endothelium injury induced by DVT through increasing eNOS in rats, which provides a potential drug for treatment of venous thrombosis.

[Tanshinone IIA alleviates monocrotaline-induced pulmonary hypertension in rats through the PI3K/Akt-eNOS signaling pathway].

OBJECTIVE: To explore the therapeutic mechanism of tanshinone IIA in the treatment of pulmonary arterial hypertension (PAH) in rats. METHODS: A total of 100 male SD rats were randomized into 5 groups (n=20), and except for those in the control group with saline injection, all the rats were injected with monocrotaline (MCT) on the back of the neck to establish models of pulmonary hypertension. Two weeks after the injection, the rat models received intraperitoneal injections of tanshinone IIA (10 mg/kg), phosphatidylinositol 3 kinase (PI3K) inhibitor (1 mg/kg), both tanshinone IIA and PI3K inhibitor, or saline (model group) on a daily basis. After 2 weeks of treatment, HE staining and α-SMA immunofluorescence staining were used to evaluate the morphology of the pulmonary vessels of the rats. The phosphorylation levels of PI3K, protein kinase B (PKB/Akt) and endothelial nitric oxide synthase (eNOS) in the lung tissue were determined with Western blotting; the levels of eNOS and NO were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of HE staining and α-SMA immunofluorescence staining showed that tanshinone IIA effectively inhibited MCT-induced pulmonary artery intimamedia thickening and muscularization of the pulmonary arterioles (P < 0.01). The results of Western blotting showed that treatment with tanshinone IIA significantly increased the phosphorylation levels of PI3K, Akt and eNOS proteins in the lung tissue of PAH rats; ELISA results showed that the levels of eNOS and NO were significantly decreased in the rat models after tanshinone IIA treatment (P < 0.01). CONCLUSION: Treatment with tanshinone IIA can improve MCT-induced pulmonary hypertension in rats through the PI3K/Akt-eNOS signaling pathway.

Intelligent Nanodelivery System-Generated 1 O2 Mediates Tumor Vessel Normalization by Activating Endothelial TRPV4-eNOS Signaling.

Tumor microenvironment (TME)-responsive intelligent photodynamic therapy (PDT) systems have attracted increasing interest in anticancer therapy, due to their potential to address significant and unsatisfactory therapeutic issues, such as limited tissue penetration, inevitable normal tissue damage, and excessive impaired vessels. Here, an H2 O2 -triggered intelligent LCL/ZnO PDT nanodelivery system is elaborately designed. LCL/ZnO can selectively regulate tumor-derived endothelial cells (TECs) and specifically kill tumor cells, by responding to different H2 O2 gradients in TECs and tumor cells. The LCL/ZnO is able to normalize tumor vessels, thereby resulting in decreased metastases, and ameliorating the immunosuppressive TME. Further analysis demonstrates that singlet oxygen (1 O2 )-activated transient receptor potential vanilloid-4-endothelial nitric oxide synthase signals generated in TECs by LCL/ZnO induce tumor vascular normalization, which is identified as a novel mechanism contributing to the increased ability of PDT to promote cancer therapy. In conclusion, designing an intelligent PDT nanodelivery system response to the TME, that includes both selective TECs regulation and specific tumor-killing, will facilitate the development of effective interventions for future clinical applications.

[Normalization of the ratio of nitric oxide and peroxynitrite by promoting eNOS dimer activity is a new direction for diabetic nephropathy treatment].

Diabetic nephropathy is a microvascular complication of diabetes. Its etiology involves metabolic disorder-induced endothelial dysfunction. Endothelium-derived nitric oxide (NO) plays an important role in a number of physiological processes, including glomerular filtration and endothelial protection. NO dysregulation is an important pathogenic basis of diabetic nephropathy. Hyperglycemia and dyslipidemia can lead to oxidative stress, chronic inflammation and insulin resistance, thus affecting NO homeostasis regulated by endothelial nitric oxide synthase (eNOS) and a conglomerate of related proteins and factors. The reaction of NO and superoxide (O2.-) to form peroxynitrite (ONOO-) is the most important pathological NO pathway in diabetic nephropathy. ONOO- is a hyper-reactive oxidant and nitrating agent in vivo which can cause the uncoupling of eNOS. The uncoupled eNOS does not produce NO but produces superoxide. Thus, eNOS uncoupling is a critical contributor of NO dysregulation. Understanding the regulatory mechanism of NO and the effects of various pathological conditions on it could reveal the pathophysiology of diabetic nephropathy, potential drug targets and mechanisms of action. We believe that increasing the stability and activity of eNOS dimers, promoting NO synthesis and increasing NO/ONOO- ratio could guide the development of drugs to treat diabetic nephropathy. We will illustrate these actions with some clinically used drugs as examples in the present review.

Melatonin protected against myocardial infarction injury in rats through a Sirt6-dependent antioxidant pathway.

BACKGROUND: The Sirt6, one of the members of the sirtuin family, has been regarded as a key factor in the pathogenesis of myocardial infarction (MI) through its antioxidant defense mechanisms. A previous study reported that melatonin is an antioxidant drug that can act as an agent for cardioprotection in cardiac ischemia-reperfusion (I/R) injury. However, whether melatonin could protect against cardiac remodeling after myocardial injury via the Sirt6-dependent antioxidant pathway remains unknown. OBJECTIVES: To explore the protective effects and the potential mechanisms of melatonin on MI-induced injury in rats. MATERIAL AND METHODS: A cardiac remodeling model was established through left coronary artery ligation surgery. The dose of melatonin was 10 mg/kg body weight. Four weeks after the treatment for 7 successive days, the infarct size and hemodynamic parameters were evaluated. The relative mRNA level and protein level of Sirt6 were also determined. Finally, the levels of oxidative stress, including reactive oxygen species (ROS) and superoxide dismutase (SOD), were measured, and the expression of nitric oxide (NO), inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS) and their corresponding phosphorylation were evaluated. RESULTS: After the treatment with melatonin, infarct size, the left ventricular end-diastolic diameter (LVEDd), and left ventricular end-systolic diameter (LVEDd) and minimum first derivative of developed pressure (min dP/dt) decreased, while left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS) and maximum first derivative of developed pressure (max dP/dt) increased in the melatonin-MI (MM) group compared to the placebo-MI (PM) group. Furthermore, the expressions of Sirt6, both in mRNA and protein level, were significantly increased in the MM group treated with melatonin, as compared to the melatonin-control (MC) group treated with melatonin. In addition, melatonin enhanced SOD activity and reduced ROS levels. At the same time, we observed that the eNOS/NO signaling pathways were activated. CONCLUSIONS: Melatonin improved cardiac function through the Sirt6-dependent antioxidant pathway in MI rats.

Cell penetrating peptides coupled to an endothelial nitric oxide synthase sequence alter endothelial permeability.

Delivery of cargo to cells through the use of cell-penetrating peptide (CPP) sequences is an area of rich investigation for targeted therapeutics. Specific to the endothelium, the layer of cells that cover every blood vessel in the body, the loss or alteration of a key enzyme, endothelial nitric oxide synthase (eNOS), is known to contribute to endothelial health during severe, infectious challenge. While the beneficial effects of eNOS are often thought to be mediated through the generation of nitric oxide, some protection is theorized to be through eNOS binding to regulatory pathways via a pentabasic RRKRK motif. We hypothesized that delivery of the eNOS-RRKRK peptide sequence using common CPPs would allow protection against gram-negative lipopolysaccharide (LPS). Combination of the eNOS-RRKRK sequence to the CPP antennapedia (AP) reduced the impact of LPS-induced permeability in cultured human microvascular endothelial cells (HMVECs) as measured by transendothelial electrical resistance (TEER). There was also a modest reduction in cytokine production, however it was observed that AP alone significantly impaired LPS-induced endothelial permeability and cytokine production. In comparison, the CPP trans-activator of transcription (TAT) did not significantly alter endothelial inflammation by itself. When TAT was coupled to the eNOS-RRKRK sequence, protection against LPS-induced permeability was still demonstrated, however cytokine production was not reduced. These data demonstrate that the RRKRK sequence of eNOS can offer some NO-independent protection against LPS-mediated endothelial inflammation, however the degree of protection is highly dependent on the type of CPP utilized for cargo delivery.

Germline GATA2 variant disrupting endothelial eNOS function and angiogenesis can be restored by c-Jun/AP-1 upregulation.

GATA2 is a transcription factor with key roles in hematopoiesis. Germline GATA2 gene variants have been associated with several inherited and acquired hematologic disorders, including myelodysplastic syndromes. Among the spectrum of GATA2 deficiency- associated manifestations thrombosis has been reported in 25% of patients, but the mechanisms are unknown. GATA2 was shown to be involved in endothelial nitric oxide synthase (eNOS) regulation and vascular development. We assessed eNOS expression and angiogenesis in patients with GATA2 deficiency. Platelets and blood outgrowth endothelial cells (BOEC) from GATA2 variant carriers showed impaired NO production and reduction of eNOS mRNA and protein expression and of eNOS activity. GATA2 binding to the eNOS gene was impaired in BOEC from GATA2-deficient patients, differently from control BOEC. GATA2 deficiency BOEC showed also defective angiogenesis, which was completely restored by treatment with the NO-donor Snitroso- N-acetylpenicillamine (SNAP). Atorvastatin, but not resveratrol, largely restored eNOS expression, NO biosynthesis and neoangiogenesis in GATA2-deficient BOEC by a mechanism involving increased expression of the eNOS transcription factor AP-1/c-JUN, replacing GATA2 when the latter is inactive. Our results unravel a possible thrombogenic mechanism of GATA2 mutations, definitely establish the regulation of eNOS by GATA2 in endothelial cells and show that endothelial angiogenesis is strictly dependent on the eNOS/NO axis. Given the ability of atorvastatin to restore NO production and angiogenesis by GATA2-deficient endothelial cells, the preventive effect of atorvastatin on thrombotic events and possibly on other clinical manifestations of the syndrome related to deranged angiogenesis should be explored in patients with GATA2 deficiency in an ad hoc designed clinical trial.

Modulation of Nitric Oxide Bioavailability Attenuates Ischemia-Reperfusion Injury in Type II Diabetes.

BACKGROUND: Diabetes mellitus increases the susceptibility of free tissue transplantations to ischemia-reperfusion injury. The aim of this study was to enhance nitric oxide (NO) bioavailability through exogenous NO synthase and the substrate L-arginine to attenuate ischemia reperfusion-induced alterations in a type 2 diabetes rodent model. MATERIAL AND METHODS: Sixty-four Wistar rats were divided into 8 experimental groups. Type 2 diabetes was established over 3 months with a combination of a high-fat diet and streptozotocin. A vascular pedicle isolated rat skin flap model that underwent 3 h of ischemia was used. At 30 min before ischemia, normal saline, endothelial NOSs (eNOSs), inducible NOSs, neuronal NOSs (1 and 2 IU), and L-arginine (50 mg/kg body weight) were administered by intravenous infusion alone or in combination. Ischemia-reperfusion-induced alterations were measured 5 days after the operation. RESULTS: The three isoforms of NOS significantly increased the flap vitality rate (VR) between 20% and 28% as compared to the control group (3%). Sole L-arginine administration increased the VR to 33%. The combination of L-arginine with NOS resulted in a further increase in flap VRs (39%-50%). Best results were achieved with the combination of eNOS and L-arginine (50%). An increase in enzyme dosage led to decreased VRs in all NOS isoforms alone and even in combination with L-arginine. CONCLUSION: Modulation of NO bioavailability through the exogenous application of NOSs and L-arginine significantly attenuated ischemia-reperfusion-induced alterations in a type 2 diabetic skin flap rat model. The combination of enzyme and substrate result in the highest VRs. Higher enzyme dosage seems to be less effective. This pharmacological preconditioning could be an easy and effective interventional strategy to support the conversion of L-arginine to NO in ischemic and in type 2 diabetic conditions.

Exercise hormone irisin mitigates endothelial barrier dysfunction and microvascular leakage-related diseases.

Increased microvascular leakage is a cardinal feature of many critical diseases. Regular exercise is associated with improved endothelial function and reduced risk of cardiovascular disease. Irisin, secreted during exercise, contributes to many health benefits of exercise. However, the effects of irisin on endothelial function and microvascular leakage remain unknown. In this study, we found that irisin remarkably strengthened endothelial junctions and barrier function via binding to integrin αVß5 receptor in LPS-treated endothelial cells. The beneficial effect of irisin was associated with suppression of the Src-MLCK-ß-catenin pathway, activation of the AMPK-Cdc42/Rac1 pathway, and improvement of mitochondrial function. In preclinical models of microvascular leakage, exogenous irisin improved pulmonary function, decreased lung edema and injury, suppressed inflammation, and increased survival. In ARDS patients, serum irisin levels were decreased and inversely correlated with disease severity and mortality. In conclusion, irisin enhances endothelial barrier function and mitigates microvascular leakage-related diseases.

Characterisation of the vasodilation effects of DHA and EPA, n-3 PUFAs (fish oils), in rat aorta and mesenteric resistance arteries.

BACKGROUND AND PURPOSE: Increasing evidence suggests that the omega-3 polyunsaturated acids (n-3 PUFA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), are beneficial to cardiovascular health, promoting relaxation of vascular smooth muscle cells and vasodilation. Numerous studies have attempted to study these responses, but to date there has not been a systematic characterisation of both DHA and EPA mediated vasodilation in conduit and resistance arteries. Therefore, we aimed to fully characterise the n-3 PUFA-induced vasodilation pathways in rat aorta and mesenteric artery. METHODS: Wire myography was used to measure the vasomotor responses of freshly dissected rat mesenteric artery and aorta. Arteries were pre-constricted with U46619 and cumulative concentrations of either DHA or EPA (10 nM-30 µM) were added. The mechanisms by which n-3 PUFA relaxed arteries were investigated using inhibitors of vasodilator pathways, which include: nitric oxide synthase (NOS; L-NAME), cycloxygenase (COX; indomethacin), cytochrome P450 epoxygenase (CYP450; clotrimazole); and calcium-activated potassium channels (KCa), SKCa (apamin), IKCa (TRAM-34) and BKCa (paxilline). RESULTS: Both DHA- and EPA-induced relaxations were partially inhibited following endothelium removal in rat mesenteric arteries. Similarly, in aorta EPA-induced relaxation was partially suppressed due to endothelium removal. CYP450 also contributed to EPA-induced relaxation in mesenteric artery. Inhibition of IKCa partially attenuated DHA-induced relaxation in aorta and mesenteric artery along with EPA-induced relaxation in mesenteric artery. Furthermore, this inhibition of DHA- and EPA-induced relaxation was increased following the additional blockade of BKCa in these arteries. CONCLUSIONS: This study provides evidence of heterogeneity in the vasodilation mechanisms of DHA and EPA in different vascular beds. Our data also demonstrates that endothelium removal has little effect on relaxations produced by either PUFA. We demonstrate IKCa and BKCa are involved in DHA-induced relaxation in rat aorta and mesenteric artery; and EPA-induced relaxation in rat mesenteric artery only. CYP450 derived metabolites of EPA may also be involved in BKCa dependent relaxation. To our knowledge this is the first study indicating the involvement of IKCa in n-3 PUFA mediated relaxation.

Effects of paracetamol on NOS, COX, and CYP activity and on oxidative stress in healthy male subjects, rat hepatocytes, and recombinant NOS.

Paracetamol (acetaminophen) is a widely used analgesic drug. It interacts with various enzyme families including cytochrome P450 (CYP), cyclooxygenase (COX), and nitric oxide synthase (NOS), and this interplay may produce reactive oxygen species (ROS). We investigated the effects of paracetamol on prostacyclin, thromboxane, nitric oxide (NO), and oxidative stress in four male subjects who received a single 3 g oral dose of paracetamol. Thromboxane and prostacyclin synthesis was assessed by measuring their major urinary metabolites 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F(1α), respectively. Endothelial NO synthesis was assessed by measuring nitrite in plasma. Urinary 15(S)-8-iso-prostaglanding F(2α) was measured to assess oxidative stress. Plasma oleic acid oxide (cis-EpOA) was measured as a marker of cytochrome P450 activity. Upon paracetamol administration, prostacyclin synthesis was strongly inhibited, while NO synthesis increased and thromboxane synthesis remained almost unchanged. Paracetamol may shift the COX-dependent vasodilatation/vasoconstriction balance at the cost of vasodilatation. This effect may be antagonized by increasing endothelial NO synthesis. High-dosed paracetamol did not increase oxidative stress. At pharmacologically relevant concentrations, paracetamol did not affect NO synthesis/bioavailability by recombinant human endothelial NOS or inducible NOS in rat hepatocytes. We conclude that paracetamol does not increase oxidative stress in humans.

Intrathecal morphine remotely preconditions the heart via a neural pathway.

Central opioid receptor activation triggers cardioprotection against ischemia reperfusion injury, independent of peripheral opioid receptor activity. Using a rodent model of myocardial ischemia reperfusion injury with infarct size as the primary outcome, we tested the hypothesis that spinal opioids confer this beneficial effect via a neural pathway. Intrathecal morphine reduced the infarct size compared with control (23% ± 7% vs. 58% ± 3%, respectively, P < 0.01). Prior antagonism of the autonomic pathway, and the receptors for bradykinin, calcitonin gene-related peptide, and the KATP channel, respectively, abolished this cardioprotection (54% ± 13%, 52% ± 10%, 56% ± 9%, and 49% ± 8%, respectively, P < 0.05). In a second set of experiments, we demonstrated that the increased expression of myocardial phosphorylated-Akt and endothelial nitric oxide synthase induced by intrathecal morphine was blocked by prior administration of hexamethonium. These findings support the notion that spinal opioid receptors stimulate a neural pathway that uses nonopioid neurotransmitters to confer cardioprotection from ischemia reperfusion injury. The use of intrathecal morphine for this purpose has potential clinical application, and it is already being used in the perioperative period to provide prolonged analgesia.