Danhong Injection Attenuates High-Fat-Induced Atherosclerosis and Macrophage Lipid Accumulation by Regulating the PI3K/AKT Insulin Pathway.

BACKGROUND AND AIMS: High-fat diet (HFD) is reported to induce atherosclerosis and insulin resistance. Macrophage lipid accumulation has been implicated as key mediators during the development of HFD-induced atherosclerosis. Traditional Chinese formula, which has long been used to improve disorder of glucose and lipid metabolism of patients, is now gradually being used as complementary therapy. This study aimed to investigate the effect of Danhong injection (DHI), a Chinese medicine used for the treatment of coronary artery disease, on atherosclerosis and its underlying mechanisms. METHODS AND RESULTS: We observed the effects of DHI on HFD-induced atherosclerosis in a mice model, macrophage lipid accumulation in an ox-LDL-stimulated macrophage model, and the role of PI3K/AKT insulin pathway in the process of DHI ameliorating atherosclerosis. The data demonstrated that DHI attenuated atherosclerosis by ameliorating blood lipids, reducing the atherosclerotic index and atherosclerotic plaque area in HFD-induced atherosclerotic mice, and inhibiting TC levels in an ox-LDL-induced macrophage model. By estimating the levels of serum insulin resistance-related indexes and protein expression of GLUT-4, DHI treatment dramatically inhibited the levels of fasting serum NEFA and fasting serum insulin and promoted the protein expression of GLUT-4 in aortas of the HFD-induced atherosclerotic mice. Moreover, according to the hints provided by microarray-based transcriptional profiling, the results demonstrated that DHI treatment also promoted the activation of PI3K/AKT insulin signaling pathway induced by IRS-1 in aortas of HFD-induced atherosclerotic mice. Furthermore, in an ox-LDL-induced macrophage model, the activation of PI3k/AKT signaling pathway also effectively functioned in the process of DHI inhibiting macrophage lipid accumulation. CONCLUSIONS: These results highlight that DHI treatment attenuates atherosclerosis and macrophage lipid accumulation by promoting the activation of PI3K/AKT insulin signaling pathway. It provides new insights into the molecular mechanism of DHI and its therapeutic potential in the treatment of atherosclerosis.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Simvastatin Does Not Affect Nitric Oxide Generation Increased by Sesame Oil in Obese Zucker Rats.

Current treatments for cardiovascular and obesity-associated diseases, such as statin therapy, may be associated with several side effects. Products from food sources with polyphenolic compounds may represent promising agents in the treatment of cardiovascular and metabolic diseases with minimal side effects. Thus, we aimed to study the effect of sesame oil and simvastatin treatment on plasma lipid profile, nitric oxide generation, and oxidative load in obese Zucker rats. 12-week-old male Zucker rats were divided into the control and sesame oil- (1.25 ml/kg/day) treated Zucker lean groups, the control and sesame oil (1.25 ml/kg/day), or simvastatin (15 mg/kg/day) together with sesame oil-treated Zucker fa/fa groups, n = 6 in each group. The treatment lasted for 6 weeks. Sesame oil composition and plasma lipid profile were analyzed. Nitric oxide synthase (NOS) activity, endothelial NOS (eNOS), phosphorylated eNOS, and inducible NOS (iNOS) protein expressions were determined in the left ventricle and aorta. Oxidative load, measured as conjugated diene (CD) and thiobarbituric acid reactive substance (TBARS) concentrations, was detected in the liver. Neither sesame oil nor cotreatment with simvastatin affected plasma lipid profile in Zucker fa/fa rats. Sesame oil and similarly cotreatment with simvastatin markedly increased NOS activity and phosphorylated eNOS protein expressions in the left ventricle and aorta of Zucker fa/fa rats. There were no changes in eNOS and iNOS protein expressions within the groups and tissues investigated. Hepatic CD concentration was higher in Zucker fa/fa comparing Zucker lean rats, and sesame oil treatment decreased it significantly. Interestingly, this decrease was not seen after cotreatment with simvastatin. In conclusion, phosphorylation of eNOS and decreased oxidative load may significantly contribute to increase in total NOS activity with potential beneficial properties. Interestingly, simvastatin did not affect NO generation already increased by sesame oil in obese Zucker rats.

Quercetin-Induced AMP-Activated Protein Kinase Activation Attenuates Vasoconstriction Through LKB1-AMPK Signaling Pathway.

The vascular tone plays an important role in blood pressure and flow. It is influenced by the contraction of vascular smooth muscle cells (VSMCs), which in turn is regulated by the balance between the myosin light chain kinase (MLCK) and the phosphorylated myosin light chain (p-MLC). Quercetin is a common flavonoid which is found in many fruits and red wine. Although quercetin has been widely reported to be involved in cell proliferation, migration, and apoptosis in VSMCs, it has not yet been demonstrated whether quercetin is related to vasocontraction, a function regulated by the AMP-activated protein kinase (AMPK) signaling pathway. Accordingly, the aim of this study is to investigate the molecular mechanism through which the quercetin-activated LKB1-AMPK signaling pathway regulates the contraction of VSMCs. In cultured VSMCs, quercetin activated AMPK in a dose- and time-dependent manner. Quercetin inhibited the phenylephrine (PE)-induced expression of MLCK and p-MLC through the LKB1-AMPK signaling pathway and decreased the mRNA level of MLCK. Adenovirus-AMPK DN α1 and AMPK DN α2-transduced VSMCs displayed higher p-MLC expression. Moreover, quercetin inhibited the PE-mediated contraction in rat aorta. These data suggest that the quercetin-activated LKB1-AMPK signaling pathway regulates VSMC contraction by inhibiting MLCK and p-MLC; hence, it may be a therapeutic intervention for the treatment of cardiovascular disorders such as atherosclerosis and hypertension.

Effects of tryptophan-containing peptides on angiotensin-converting enzyme activity and vessel tone ex vivo and in vivo.

PURPOSE: Over-activation of the renin-angiotensin axis and worsening of vascular function are critical contributors to the development of hypertension. Therefore, inhibition of angiotensin-converting enzyme (ACE), a key factor of the renin-angiotensin axis, is a first line treatment of hypertension. Besides pharmaceutical ACE inhibitors, some natural peptides have been shown to exert ACE-inhibiting properties with antihypertensive effects and potentially beneficial effects on vascular function. In this study, the ACE-inhibiting potential and effects on vascular function of tryptophan-containing peptides were evaluated. METHODS: The ACE inhibitory action and stability of tryptophan-containing peptides was tested in endothelial cells-a major source of whole body ACE activity. Furthermore, the efficacy of peptides on vascular ACE activity, as well as vessel tone was assessed both ex vivo and in vivo. RESULTS: In human umbilical vein endothelial cells (HUVEC), isoleucine-tryptophan (IW) had the highest ACE inhibitory efficacy, followed by glutamic acid-tryptophan (EW) and tryptophan-leucine (WL). Whereas none of the peptides affected basal vessel tone (rat aorta), angiotensin I-induced vasoconstriction was blocked. IW effectively inhibited aortic ACE activity ex vivo taken from SHRs after 14-weeks of oral treatment with IW. Furthermore, IW treated SHRs showed better endothelium-dependent vessel relaxation compared to placebo. CONCLUSION: This study shows strong ACE-inhibiting effects of IW, EW and WL in HUVECs and aorta. The peptides effectively counteract angiotensin-induced vasoconstriction and preserve endothelium-dependent vessel relaxation. Thus, tryptophan-containing peptides and particularly IW may serve as innovative food additives with the goal of protection from angiotensin II-induced worsening of vascular function.

From the Cover: Lung-Specific Overexpression of Constitutively Active IKK2 Induces Pulmonary and Systemic Inflammations but Not Hypothalamic Inflammation and Glucose Intolerance.

The lung is constantly exposed to ambient pollutants such as ambient fine particulate matter (PM2.5), making it one of the most frequent locations of inflammation in the body. Given the establishment of crucial role of inflammation in the pathogenesis of cardiometabolic diseases, pulmonary inflammation is thus widely believed to be an important risk factor for cardiometabolic diseases. However, the causality between them has not yet been well established. To determine if pulmonary inflammation is sufficient to cause adverse cardiometabolic effects, SFTPC-rtTA+/-tetO-cre+/-pROSA-inhibitor κB kinase 2(IKK2)ca+/- (LungIKK2ca) and littermate SFTPC-rtTA+/-tetO-cre-/-pROSA-IKK2ca+/- wildtype (WT) mice were fed with doxycycline diet to induce constitutively active Ikk2 (Ikk2ca) overexpression in the lung and their pulmonary, systemic, adipose, and hypothalamic inflammations, vascular function, and glucose homeostasis were assessed. Feeding with doxycycline diet resulted in IKK2ca overexpression in the lungs of LungIKK2ca but not WT mice. This induction of IKK2ca was accompanied by marked pulmonary inflammation as evidenced by significant increases in bronchoalveolar lavage fluid leukocytes, pulmonary macrophage infiltration, and pulmonary mRNA expression of tumor necrosis factor α (Tnfα) and interleukin-6 (Il-6). This pulmonary inflammation due to lung-specific overexpression of IKK2ca was sufficient to increase circulating TNFα and IL-6 levels, adipose expression of Tnfα and Il-6 mRNA, aortic endothelial dysfunction, and systemic insulin resistance. Unexpectedly, no significant alteration in hypothalamic expression of Tnfα and Il-6 mRNA and glucose intolerance were observed in these mice. Pulmonary inflammation is sufficient to induce systemic inflammation, endothelial dysfunction, and insulin resistance, but not hypothalamic inflammation and glucose intolerance.

Quercetin Attenuates Vascular Calcification through Suppressed Oxidative Stress in Adenine-Induced Chronic Renal Failure Rats.

BACKGROUND: This study investigated whether quercetin could alleviate vascular calcification in experimental chronic renal failure rats induced by adenine. METHODS: 32 adult male Wistar rats were randomly divided into 4 groups fed normal diet, normal diet with quercetin supplementation (25 mg/kg·BW/d), 0.75% adenine diet, or adenine diet with quercetin supplementation. All rats were sacrificed after 6 weeks of intervention. Serum renal functions biomarkers and oxidative stress biomarkers were measured and status of vascular calcification in aorta was assessed. Furthermore, the induced nitric oxide synthase (iNOS)/p38 mitogen activated protein kinase (p38MAPK) pathway was determined to explore the potential mechanism. RESULTS: Adenine successfully induced renal failure and vascular calcification in rat model. Quercetin supplementation reversed unfavorable changes of phosphorous, uric acid (UA) and creatinine levels, malonaldehyde (MDA) content, and superoxide dismutase (SOD) activity in serum and the increases of calcium and alkaline phosphatase (ALP) activity in the aorta (P < 0.05) and attenuated calcification and calcium accumulation in the medial layer of vasculature in histopathology. Western blot analysis showed that iNOS/p38MAPK pathway was normalized by the quercetin supplementation. CONCLUSIONS: Quercetin exerted a protective effect on vascular calcification in adenine-induced chronic renal failure rats, possibly through the modulation of oxidative stress and iNOs/p38MAPK pathway.

Effect of Bioactive Compound of Aronia melanocarpa on Cardiovascular System in Experimental Hypertension.

Aronia melanocarpa has attracted scientific interest due to its dense contents of different polyphenols. We aimed to analyse effects of Aronia melanocarpa (AME) extract on blood pressure (BP), lipid peroxidation, cytokine level, total NOS activity in the left ventricle (LV), and aorta of L-NAME-induced hypertensive rats. 12-week-old male WKY rats were assigned to the control group and groups treated with AME extract (57.90 mg/kg/day), L-NAME (40 mg/kg/day), or combination of L-NAME (40 mg/kg/day) and AME (57.90 mg/kg/day) in tap water for 3 weeks. NOS activity, eNOS protein expression, and conjugated diene (CD) concentration were determined in the LV and aorta. After 3 weeks of L-NAME treatment, BP was increased by 28% and concomitant treatment with AME reduced it by 21%. NOS activity of the LV and aorta in the L-NAME group was decreased by about 40%, while AME increased it almost on the control level. AME-induced eNOS upregulation may contribute to increase NOS activity. Moreover, AME decreased CD concentration in the LV and aorta and TNF-α and IL-6 production in the plasma were increased by L-NAME treatment. In conclusion, our results showed that active substances of Aronia melanocarpa may have a positive effect on blood pressure, NOS activity, and proinflammatory processes in L-NAME-induced hypertension.

Arginase Inhibitor 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-D-Glucoside Activates Endothelial Nitric Oxide Synthase and Improves Vascular Function.

Endothelial arginase constrains the activity of endothelial nitric oxide synthase by reducing nitric oxide bioavailability, which contributes to vascular diseases. During screening, we identified a novel compound from the rhizome of Polygonum multiflorum (Polygonaceae), 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG), which inhibited arginase activity. THSG exhibited noncompetitive inhibition of arginase II and inhibited both arginases I and II in a dose-dependent manner. THSG-dependent arginase inhibition reciprocally increased nitric oxide production and decreased reactive oxygen species generation in aortic endothelia. These effects were associated with increased dimerization of endothelial nitric oxide synthase without changes in the protein expression levels of arginase I, arginase II, or endothelial nitric oxide synthase. In vascular tension assays, when aortic vessels from wild-type mice are incubated with THSG, responses to the nitric oxide-dependent vasorelaxant acetylcholine were augmented, but responses to an nitric oxide donor, sodium nitroprusside, were not affected. On the other hand, phenylephrine-dependent vasoconstriction was significantly retarded in THSG-treated vessels. In a high-cholesterol diet-fed atherogenic model mice (ApoE-/-), THSG improved endothelial function by enhancement of the nitric oxide-cGMP pathway. Taken together, these results suggest that THSG may exert vasoprotective effects through augmentation of nitric oxide signaling by inhibiting arginase. Therefore, THSG may be useful in the treatment of vascular diseases that are derived from endothelial dysfunction, such as atherosclerosis.

Dihydroquercetin Does Not Affect Age-Dependent Increase in Blood Pressure and Angiotensin-Converting Enzyme Activity in the Aorta of Hypertensive Rats.

We analyzed changes in angiotensin-converting enzyme activity in the aorta of hypertensive SHR rats against the background of age-related BP increase (from week 7 to 14) and the effect of dihydroquercetin on BP rise and angiotensin-converting enzyme activity. Normotensive WKY rats of the same age were used as the control. BP and activity of angiotensin-converting enzyme in the aorta of SHR rats increased with age. Dihydroquercetin in doses of 100 and 300 µg/kg per day had no effect on the increase of these parameters; dihydroquercetin administered to 14-week-old WKY rats in a dose of 300 µg/kg reduced activity of the angiotensin-converting enzyme. Thus, the early (7-14 weeks) increase in BP and angiotensin-converting enzyme activity in the aorta of SHR rats was not modified by flavonoids (dihydroquercetin) in contrast to other rat strains and humans, which is indicative of specificity of hypertension mechanism in SHR rats.

Inhibiting adhesion events by Panax notoginseng saponins and Ginsenoside Rb1 protecting arteries via activation of Nrf2 and suppression of p38 - VCAM-1 signal pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Asian countries, such as China, Japan, and Korea, have witnessed a history of more than 1000 years of Panax notoginseng (Burk.) F.H. Chen's application as a famous traditional medicine for cardiovascular diseases (Zhou et al., 2004). The use of Panax notoginseng (Sanqi) was first recorded in "Bencao Gangmu", which was written by Li Shizhen, a Chinese pharmacologist of the MING dynasty, in 1578. It is included in "The Plant List" as one species of genus Panax (family Araliaceae). Panax notoginseng saponins (PNS) are the major active ingredients extracted from Panax notoginseng. AIM OF THE STUDY: This study investigated whether PNS and the active constituent Ginsenoside Rb1 inhibits adhesion events by regulating the NF-E2-related factor 2 (Nrf2) - p38 - vascular cell adhesion molecule (VCAM)-1 pathway. MATERIALS AND METHODS: The AS model rats were treated once daily with PNS (100mg/kg, i.p.) or Rb1 (40mg/kg, i.p.), and pathological changes in the aortas were observed by electron microscopy and Sudan IV staining. The serum levels of NO, superoxide dismutase (SOD) and TNF-α were measured. Upon treatment with H2O2 to induce oxidative stress, cell viability and LDH levels were measured after cells were cultured with PNS or Rb1. oxidized low density lipoprotein (oxLDL)-induced VCAM-1 and p38 protein expression and THP1 cell adhesion to ECs were assessed after treatment with PNS or Rb1. Nuclear translocation of Nrf2 and expression of its target protein heme oxygenase (HO)-1 were observed in the respective presence of PNS or Rb1. RESULTS: Upon treatment with PNS or Rb1, pathological changes observed in the aortas of AS model rats were alleviated, and an increase in serum levels of NO and SOD and a decrease in TNF-α levels were observed. In vitro treatment with PNS or Rb1 protected endothelial cells (ECs) from H2O2-mediated cytotoxicity, suppressed oxLDL-induced p38 and VCAM-1 protein expression and inhibited THP1 cell adhesion to ECs. Finally, PNS and Rb1 treatment functionally activated Nrf2 in ECs. CONCLUSIONS: Nrf2, an EC protective system, suppresses monocyte adhesion events via the inhibition of the ROS - TNF-α - p38 - VCAM-1 pathway following treatment with PNS, with Rb1 specifically playing an important role among PNS active components.

Cytochrome P450 Oxidase 2C Inhibition Adds to ω-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization.

OBJECTIVE: Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but ω-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of ω-3 LCPUFA on neovascular eye diseases. APPROACH AND RESULTS: The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of ω-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and ω-3 LCPUFA-derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of ω-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a ω-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C ω-3 LCPUFA-derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected ω-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. CONCLUSIONS: Inhibition of CYP2C activity adds to the protective effects of ω-3 LCPUFA on pathological retinal neovascularization and CNV.

Etanercept improves endothelial function via pleiotropic effects in rat adjuvant-induced arthritis.

OBJECTIVES: To determine the effect of etanercept on endothelial dysfunction and on traditional cardiovascular (CV) risk factors in the adjuvant-induced arthritis (AIA) rat model. METHODS: At the first signs of arthritis, etanercept (10 mg/kg/3 days, s.c.) or saline was administered for 3 weeks in AIA rats. Body weights and arthritis scores were monitored daily. Endothelial function was studied in aortic rings relaxed with acetylcholine (Ach) with or without inhibitors of nitric oxide synthase (NOS), cyclo-oxygenase (COX-2), arginase, endothelium-derived hyperpolarizing factor and superoxide anions (O2 (-)°) production. Aortic expression of endothelial nitic oxide synthase (eNOS), Ser1177-phospho-eNOS, COX-2, arginase-2, p22(phox) and p47(phox) was evaluated by western blotting analysis. Blood pressure, heart rate and blood levels of triglycerides, cholesterol and glucose were measured. RESULTS: Etanercept significantly reduced arthritis score (P < 0.001). It improved Ach-induced relaxation (P < 0.05) as a result of increased NOS activity, decreased COX-2/arginase activities and decreased O2 (-)° production. These functional effects relied on increased eNOS expression and phosphorylation, and decreased COX-2, arginase-2 and p22(phox) expressions. No correlation was found between arthritis score and Ach-induced relaxation. The treatment did not change triglycerides, cholesterol and glucose levels, but significantly increased systolic blood pressure and heart rate (P < 0.05). CONCLUSION: Our data demonstrated that efficient dosage of etanercept on inflammatory symptoms improved endothelial function in AIA. This beneficial effect on endothelial function is disconnected from its impact on CV risk factors and relates to pleiotropic effects of etanercept on endothelial pathways. These results suggest that etanercept could be a good choice for patients with rheumatoid arthritis at high risk of CV events.

Plasmalogen modulation attenuates atherosclerosis in ApoE- and ApoE/GPx1-deficient mice.

BACKGROUND AND AIM: We previously reported a negative association of circulating plasmalogens (phospholipids with proposed atheroprotective properties) with coronary artery disease. Plasmalogen modulation was previously demonstrated in animals but its effect on atherosclerosis was unknown. We assessed the effect of plasmalogen enrichment on atherosclerosis of murine models with differing levels of oxidative stress. METHODS AND RESULTS: Six-week old ApoE- and ApoE/glutathione peroxidase-1 (GPx1)-deficient mice were fed a high-fat diet with/without 2% batyl alcohol (precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids showed that batyl alcohol supplementation to ApoE- and ApoE/GPx1-deficient mice increased the total plasmalogen levels in both plasma and heart. Oxidation of plasmalogen in the treated mice was evident from increased level of plasmalogen oxidative by-product, sn-2 lysophospholipids. Atherosclerotic plaque in the aorta was reduced by 70% (P = 5.69E-07) and 69% (P = 2.00E-04) in treated ApoE- and ApoE/GPx1-deficient mice, respectively. A 40% reduction in plaque (P = 7.74E-03) was also seen in the aortic sinus of only the treated ApoE/GPx1-deficient mice. Only the treated ApoE/GPx1-deficient mice showed a decrease in VCAM-1 staining (-28%, P = 2.43E-02) in the aortic sinus and nitrotyrosine staining (-78%, P = 5.11E-06) in the aorta. CONCLUSION: Plasmalogen enrichment via batyl alcohol supplementation attenuated atherosclerosis in ApoE- and ApoE/GPx1-deficient mice, with a greater effect in the latter group. Plasmalogen enrichment may represent a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk, particularly under conditions of elevated oxidative stress and inflammation.

The Green Tea Component (-)-Epigallocatechin-3-Gallate Sensitizes Primary Endothelial Cells to Arsenite-Induced Apoptosis by Decreasing c-Jun N-Terminal Kinase-Mediated Catalase Activity.

The green tea component (-)-epigallocatechin-3-gallate (EGCG) has been shown to sensitize many different types of cancer cells to anticancer drug-induced apoptosis, although it protects against non-cancerous primary cells against toxicity from certain conditions such as exposure to arsenic (As) or ultraviolet irradiation. Here, we found that EGCG promotes As-induced toxicity of primary-cultured bovine aortic endothelial cells (BAEC) at doses in which treatment with each chemical alone had no such effect. Increased cell toxicity was accompanied by an increased condensed chromatin pattern and fragmented nuclei, cleaved poly(ADP-ribose) polymerase (PARP), activity of the pro-apoptotic enzymes caspases 3, 8 and 9, and Bax translocation into mitochondria, suggesting the involvement of an apoptotic signaling pathway. Fluorescence activated cell sorting analysis revealed that compared with EGCG or As alone, combined EGCG and As (EGCG/As) treatment significantly induced production of reactive oxygen species (ROS), which was accompanied by decreased catalase activity and increased lipid peroxidation. Pretreatment with N-acetyl-L-cysteine or catalase reversed EGCG/As-induced caspase activation and EC toxicity. EGCG/As also increased the phosphorylation of c-Jun N-terminal kinase (JNK), which was not reversed by catalase. However, pretreatment with the JNK inhibitor SP600125 reversed all of the observed effects of EGCG/As, suggesting that JNK may be the most upstream protein examined in this study. Finally, we also found that all the observed effects by EGCG/As are true for other types of EC tested. In conclusion, this is firstly to show that EGCG sensitizes non-cancerous EC to As-induced toxicity through ROS-mediated apoptosis, which was attributed at least in part to a JNK-activated decrease in catalase activity.

The antiatherogenic effect of bixin in hypercholesterolemic rabbits is associated to the improvement of lipid profile and to its antioxidant and anti-inflammatory effects.

Hypercholesterolemia and oxidative stress have been implicated in the pathophysiology of atherosclerosis and coronary artery disease. We investigated whether the carotenoid bixin (BIX) may reduce oxidative damage, inflammatory response, and the atherosclerotic lesion induced by hypercholesterolemia in rabbits. Rabbits received regular chow (control) or a hypercholesterolemic diet (0.5% cholesterol) alone or supplemented with BIX (10, 30 or 100 mg/kg body weight, b.w.) or simvastatin (15 mg/kg b.w.) for 60 days. Treatment with BIX or simvastatin reduced the atherosclerotic lesions in cholesterol-fed rabbits (up to 55 and 96% reduction, respectively). This protective effect of BIX was accompanied by decrease in the levels of tumor necrosis factor alpha by 15%, interleukin 6 by 19%, lipid peroxidation by 60%, non-high-density lipoprotein cholesterol (non-HDL-C) by 37%, and triglycerides by 41%. BIX increased by 160% the HDL-C levels and decreased by 67% the atherogenic index of hypercholesterolemic rabbits. In atherosclerotic rabbits, the non-protein thiol groups content and the activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione reductase, and thioredoxin reductase were increased in the aortic tissue, whereas paraoxonase activity was reduced in the serum. All these changes were completely prevented by BIX or simvastatin treatment. These results demonstrate that BIX reduces the extent of atherosclerotic lesions and this effect was associated with the decrease in oxidative stress, inflammatory response, and improvement of dyslipidemia, which were most effectively controlled after treatment with 10-30 mg BIX/kg b.w. BIX consumption may, therefore, be an adjuvant to prevent atherosclerosis reducing risk factors for coronary diseases.

Camel thorn extract reduces activity of angiotensin-converting enzyme in rat aorta increased during aging and treatment with NO-synthase inhibitor.

We studied the effect of camel thorn extract on activity of angiotensin-converting enzyme in rat aorta increased in animals during aging or treatment with NO-synthase inhibitor. Intake of camel thorn extract with drinking water reduced activity of angiotensin-converting enzyme; the effect increased with increasing the dose of the extract. Angiotensin-converting enzyme activity in older rats and animals treated with NO-synthase inhibitor decreased to the values observed in young control rats at extract concentration of 0.2%. Comparison of the effects of camel thorn extract with those of flavonoid taxifolin showed that the extract was not inferior to taxifolin in preventing the early stages of aortic atherosclerosis caused by increased activity of angiotensin-converting enzyme.

Sodium tanshinone IIA silate inhibits high glucose-induced vascular smooth muscle cell proliferation and migration through activation of AMP-activated protein kinase.

The proliferation of vascular smooth muscle cells may perform a crucial role in the pathogenesis of diabetic vascular disease. AMPK additionally exerts several salutary effects on vascular function and improves vascular abnormalities. The current study sought to determine whether sodium tanshinone IIA silate (STS) has an inhibitory effect on vascular smooth muscle cell (VSMC) proliferation and migration under high glucose conditions mimicking diabetes without dyslipidemia, and establish the underlying mechanism. In this study, STS promoted the phosphorylation of AMP-activated protein kinase (AMPK) at T172 in VSMCs. VSMC proliferation was enhanced under high glucose (25 mM glucose, HG) versus normal glucose conditions (5.5 mM glucose, NG), and this increase was inhibited significantly by STS treatment. We utilized western blotting analysis to evaluate the effects of STS on cell-cycle regulatory proteins and found that STS increased the expression of p53 and the Cdk inhibitor, p21, subsequent decreased the expression of cell cycle-associated protein, cyclin D1. We further observed that STS arrested cell cycle progression at the G0/G1 phase. Additionally, expression and enzymatic activity of MMP-2, translocation of NF-κB, as well as VSMC migration were suppressed in the presence of STS. Notably, Compound C (CC), a specific inhibitor of AMPK, as well as AMPK siRNA blocked STS-mediated inhibition of VSMC proliferation and migration. We further evaluated its potential for activating AMPK in aortas in animal models of type 2 diabetes and found that Oral administration of STS for 10 days resulted in activation of AMPK in aortas from ob/ob or db/db mice. In conclusion, STS inhibits high glucose-induced VSMC proliferation and migration, possibly through AMPK activation. The growth suppression effect may be attributable to activation of AMPK-p53-p21 signaling, and the inhibitory effect on migration to the AMPK/NF-κB signaling axis.

Rho kinase inhibition activity of pinocembrin in rat aortic rings contracted by angiotensin II.

AIM: To investigate the effects of pinocembrin on angiotensin II (Ang II)-induced vascular contraction, and to explore its molecular mechanism of actions. METHODS: The isometric vascular tone was measured in rat thoracic aortic rings with denuded endothelium. Phosphorylation level of myosin phosphatase target unit 1 (MYPT1), and protein levels of Rho kinase 1 (ROCK1, ROKß or p160ROCK) and angiotensin II type-1 receptor (AT1R) were determined by Western blot analysis. RESULTS: Pinocembrin produced a relaxant effect on endothelium-denuded aortic rings contracted by Ang II (100 nmol·L(-1)) in a dose-dependent manner. In endothelium-denuded aortic rings stimulated by Ang II, pretreatment with pinocembrin (25 and 100 µmol·L(-1)) for 20 min significantly attenuated MYPT1 phosphorylation and ROCK1 protein levels. Meanwhile, the protein level of AT1R in response to Ang II was not affected by pinocembrin in rat aortic rings. CONCLUSION: These findings indicate that pinocembrin inhibits vasoconstriction induced by Ang II in rat endothelium-denuded aortic rings, and the mechanism at least in part, is due to the blockade of the RhoA/ROCK pathway.

Effect of nitric oxide on naphthoquinone toxicity in endothelial cells: role of bioenergetic dysfunction and poly (ADP-ribose) polymerase activation.

When produced at physiological levels, reactive oxygen species (ROS) can act as signaling molecules to regulate normal vascular function. Produced under pathological conditions, ROS can contribute to the oxidative damage of cellular components (e.g., DNA and proteins) and trigger cell death. Moreover, the reaction of superoxide with nitric oxide (NO) produces the strong oxidant peroxynitrite and decreases NO bioavailability, both of which may contribute to activation of cell death pathways. The effects of ROS generated from the 1,4-naphthoquinones alone and in combination with NO on the activation status of poly(ADP-ribose) polymerase (PARP) and cell viability were examined. Treatment with redox cycling quinones activates PARP, and this stimulatory effect is attenuated in the presence of NO. Mitochondria play a central role in cell death signaling pathways and are a target of oxidants. We show that simultaneous exposure of endothelial cells to NO and ROS results in mitochondrial dysfunction, ATP and NAD(+) depletion, and cell death. Alone, NO and ROS have only minor effects on cellular bioenergetics. Further, PARP inhibition does not attenuate reduced cell viability or mitochondrial dysfunction. These results show that concomitant exposure to NO and ROS impairs energy metabolism and triggers PARP-independent cell death. While superoxide-mediated PARP activation is attenuated in the presence of NO, PARP inhibition does not modify the loss of mitochondrial function or adenine and pyridine nucleotide pools and subsequent bioenergetic dysfunction. These findings suggest that the mechanisms by which ROS and NO induce endothelial cell death are closely linked to the maintenance of mitochondrial function and not overactivation of PARP.