Biosynthesis of D-danshensu from L-DOPA using engineered Escherichia coli whole cells.

D-Danshensu (D-DSS), a traditional Chinese medicine, is used to treat cardiovascular and cerebrovascular diseases. However, current isolation protocols for D-DSS both natural and synthetic are not ideal; therefore, in this study, we have developed a whole-cell biotransformation method to produce D-DSS from L-DOPA. This was done by co-expressing L-amino acid deaminase (aadL), D-lactate dehydrogenase (ldhD), and glucose dehydrogenase (gdh). To begin to optimize the production of D-DSS, varying copy number plasmids were used to express each of the required genes. The resulting strain, Escherichia coli ALG7, which strongly overexpressed aadL, ldhD, and weakly overexpressed gdh, yielded a 378% increase in D-DSS production compared to E. coli ALG1. Furthermore, the optimal reaction conditions for the production of D-DSS were found to be a pH of 7.5, temperature at 35 °C, and 50 g/L wet cells for 12 h. Under these optimized conditions, the D-DSS amount achieved 119.1 mM with an excellent ee (> 99.9%) and a productivity of 9.9 mM/h.

(-)-Epicatechin induced reversal of endothelial cell aging and improved vascular function: underlying mechanisms.

The consumption of cocoa products rich in (-)-epicatechin is associated with reduced cardiovascular risk and improved vascular function. However, little is known about (-)-epicatechin's effects on aged endothelium. In order to characterize the health restoring effects of (-)-epicatechin on aged endothelium and identify the underlying mechanisms, we utilized high passage number (i.e. aged) bovine coronary artery endothelial cells and aortas of 3 and 18 month old rats. We evaluated cell senescence (ß-galactosidase), nitric oxide (NO) production through the endothelial nitric oxide synthase pathway, mitochondria related endpoints, citrate synthase activity and vascular relaxation. Cells were treated with water or (-)-epicatechin (1 µM) for 48 h and rats orally with either water or (-)-epicatechin (1 mg kg-1 day-1) for 15 days. Senescence associated ß-galactosidase levels doubled in aged cells while those treated with (-)-epicatechin only evidenced an ∼40% increase. NO levels in cells decreased by ∼33% with aging and (-)-epicatechin normalized them. Endothelial nitric oxide synthase phosphorylation levels paralleled these results. Aging increased total protein and synthase acetylation levels and (-)-epicatechin partially restored them to those of young cells by stimulating sirtuin-1 binding to the synthase. Phosphorylated sirtuin-1, mitofilin, oxidative phosphorylation complexes and transcriptional factor for mitochondria were reduced by ∼40% with aging and were restored by (-)-epicatechin. (-)-Epicatechin enhanced acetylcholine induced aged aorta vasodilation and stimulated NO levels while reducing blood pressure. In conclusion, (-)-epicatechin reverses endothelial cell aging and restores key control elements of vascular function. These actions may partly explain the epidemiological evidence for the beneficial effects of cocoa consumption on the incidence of cardiac and vascular diseases.

Omega-3 Polyunsaturated Fatty Acids Protect Against Cigarette Smoke-Induced Oxidative Stress and Vascular Dysfunction.

In cigarette smokers endothelial dysfunction, measured by flow-mediated dilation (FMD), precedes cardiovascular disease (CVD) and can be improved by supplementation with n - 3 polyunsaturated fatty acids (PUFAs). We developed a mouse model of cigarette smoke (CS)-induced endothelial dysfunction that resembles impaired FMD observed in human cigarette smokers and investigated the mechanism by which n - 3 PUFAs mediate vasoprotection. We hypothesized that loss of nitric oxide (NO)-dependent vasodilation in CS-exposed mice would be prevented by dietary n - 3 PUFAs via a decrease in oxidative stress. C57BL/6 mice were fed a chow or n - 3 PUFA diet for 8 weeks and then exposed to mainstream CS or filtered air for 5 days, 2 h/day. Mesenteric arterioles were preconstricted with U46619 and dilated by stepwise increases in pressure (0-40 mmHg), resulting in increases in flow, ± inhibitor of NO production or antioxidant, Tempol. Markers of oxidative stress were measured in lung and heart. CS-exposed mice on a chow diet had impaired FMD, resulting from loss of NO-dependent dilation, compared with air exposed mice. Tempol restored FMD by normalizing NO-dependent dilation and increasing NO-independent dilation. CS-exposed mice on the n - 3 PUFA diet had normal FMD, resulting from a significant increase in NO-independent dilation, compared with CS-exposed mice on a chow diet. Furthermore, n - 3 PUFAs decreased two CS-induced markers of oxidative stress, 8-epiprostaglandin-F2α levels and heme oxygenase-1 mRNA, and significantly attenuated CS-induced cytochrome P4501A1 mRNA expression. These data demonstrate that dietary n - 3 PUFAs can protect against CS-induced vascular dysfunction via multiple mechanisms, including increasing NO-independent vasodilation and decreasing oxidative stress.

Serelaxin in acute heart failure: Most recent update on clinical and preclinical evidence.

Heart failure continues to be a widely prevalent disease across the world, affecting millions of Americans annually. Acute heart failure (AHF) has a substantial effect on rising healthcare costs and is one of the major causes of morbidity and mortality. The search for new drugs for symptom relief and to improve long-term outcomes in heart failure has led to development of serelaxin, a recombinant human relaxin-2 hormone. Relaxin was discovered in pregnancy, but eventually found to have a number of other physiological actions, not only in pregnancy, but also in nonpregnant women and men. The actions of serelaxin are primarily via nitric oxide, leading to the observed vasodilatory effects, and increase in renal plasma flow. It has also been found to increase expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2 and MMP-9. The antifibrotic and antiinflammatory effects of the drug also play a role in heart failure. In Phase II studies, serelaxin has shown reduction in pulmonary arterial pressure, pulmonary capillary wedge pressure, and NT-proBNP. The recently published results of the RELAX-AHF, a phase III clinical trial on serelaxin, has opened new avenues into our understanding of its effects in heart failure. The trial showed improvement in short-term dyspnea scores and 180-day mortality, but, interestingly, failed to show any improvement of the secondary endpoints of death or readmission at 60 days. Ongoing Phase III trials like RELAX-AHF-2 and RELAX-AHF-ASIA would explain these data better and improve understanding of the use of serelaxin in clinical practice. This article summarizes the most updated published preclinical and clinical study data on serelaxin, including pharmacokinetic, pharmacodynamic, safety studies in hepatic, renal impaired patients, Phase II and Phase III trials.

Omega-3 polyunsaturated fatty acids and oxygenated metabolism in atherothrombosis.

Numerous epidemiological studies and clinical trials have reported the health benefits of omega-3 polyunsaturated fatty acids (PUFA), including a lower risk of coronary heart diseases. This review mainly focuses on the effects of alpha-linolenic (ALA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids on some risk factors associated with atherothrombosis, including platelet activation, plasma lipid concentrations and oxidative modification of low-density lipoproteins (LDL). Special focus is given to the effects of marine PUFA on the formation of eicosanoids and docosanoids, and to the bioactive properties of some oxygenated metabolites of omega-3 PUFA produced by cyclooxygenases and lipoxygenases. The antioxidant effects of marine omega-3 PUFA at low concentrations and the pro-oxidant effects of DHA at high concentrations on the redox status of platelets and LDL are highlighted. Non enzymatic peroxidation end-products deriving from omega-3 PUFA such as hydroxy-hexenals, neuroketals and EPA-derived isoprostanes are also considered in relation to atherosclerosis. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Increased accumulation of the cardio-cerebrovascular disease treatment drug tanshinone in Salvia miltiorrhiza hairy roots by the enzymes 3-hydroxy-3-methylglutaryl CoA reductase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

Tanshinone is widely used for treatment of cardio-cerebrovascular diseases with increasing demand. Herein, key enzyme genes SmHMGR (3-hydroxy-3-methylglutaryl CoA reductase) and SmDXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase) involved in the tanshinone biosynthetic pathway were introduced into Salvia miltiorrhiza (Sm) hairy roots to enhance tanshinone production. Over-expression of SmHMGR or SmDXR in hairy root lines can significantly enhance the yield of tanshinone. Transgenic hairy root lines co-expressing HMGR and DXR (HD lines) produced evidently higher levels of total tanshinone (TT) compared with the control and single gene transformed lines. The highest tanshinone production was observed in HD42 with the concentration of 3.25 mg g(-1) DW. Furthermore, the transgenic hairy roots showed higher antioxidant activity than control. In addition, transgenic hairy root harboring HMGR and DXR (HD42) exhibited higher tanshinone content after elicitation by yeast extract and/or Ag(+) than before. Tanshinone can be significantly enhanced to 5.858, 6.716, and 4.426 mg g(-1) DW by YE, Ag(+), and YE-Ag(+) treatment compared with non-induced HD42, respectively. The content of cryptotanshinone and dihydrotanshinone was effectively elevated upon elicitor treatments, whereas there was no obvious promotion effect for the other two compounds tanshinone I and tanshinone IIA. Our results provide a useful strategy to improve tanshinone content as well as other natural active products by combination of genetic engineering with elicitors.

Effects of selected bioactive natural products on the vascular endothelium.

The endothelium, a highly active structure, regulates vascular homeostasis through the release of numerous vasoactive factors that control vascular tone and vascular smooth cell proliferation. A larger number of medicinal plants and their isolated chemical constituents have been shown to beneficially affect the endothelium. For example, flavonoids in black tea, green tea, and concord grape cause a vasodilation possibly through their antioxidant properties. Allicin, a by-product of the enzyme alliinase, has been proposed to be the main active metabolite and responsible for most of the biological activities of garlic, including a dose-dependent dilation on the isolated coronaries. Thymoquinone, the principal phytochemical compound found in the volatile oil of the black seed, and the hawthorn extract have also been shown to improve aging-related impairment of endothelium-dependent relaxations in animal models. In this review, the effect of some of the natural products, including Camellia sinensis (black tea and green tea), Vitis labrusca (concord grape), Allium sativum (garlic), and Nigella sativa (black seed) and Crataegus ssp (hawthorn extract), is explored. The molecular mechanisms behind these potential therapeutic effects are also discussed.

Comparison of menaquinone-4 and menaquinone-7 bioavailability in healthy women.

BACKGROUND: Vitamin K2 contributes to bone and cardiovascular health. Therefore, two vitamin K2 homologues, menaquinone-4 (MK-4) and menaquinone-7 (MK-7), have been used as nutrients by the food industry and as nutritional supplements to support bone and cardiovascular health. However, little is known about the bioavailability of nutritional MK-4. To investigate MK-4 and MK-7 bioavailability, nutritional doses were administered to healthy Japanese women. FINDINGS: Single dose administration of MK-4 (420 µg; 945 nmol) or MK-7 (420 µg; 647 nmol) was given in the morning together with standardized breakfast. MK-7 was well absorbed and reached maximal serum level at 6 h after intake and was detected up to 48 h after intake. MK-4 was not detectable in the serum of all subjects at any time point. Consecutive administration of MK-4 (60 µg; 135 nmol) or MK-7 (60 µg; 92 nmol) for 7 days demonstrated that MK-4 supplementation did not increase serum MK-4 levels. However, consecutive administration of MK-7 increased serum MK-7 levels significantly in all subjects. CONCLUSIONS: We conclude that MK-4 present in food does not contribute to the vitamin K status as measured by serum vitamin K levels. MK-7, however significantly increases serum MK-7 levels and therefore may be of particular importance for extrahepatic tissues.

Metabolism of dl-praeruptorin A in rat liver microsomes using HPLC-electrospray ionization tandem mass spectrometry.

dl-Praeruptorin A (Pd-Ia) is the major active constituent of the traditional Chinese medicine Peucedanum praeruptorum Dunn. Recently it has been identified as a novel agent in the treatment and prevention of cardiovascular diseases. Accordingly, we investigated the metabolism of Pd-Ia in rat liver microsomes. The involvement of cytochrome P450 (CYP) and CYP isoforms were identified using a CYP-specific inhibitor (SKF-525A), CYP-selective inhibitors (α-naphthoflavone, metyrapone, fluvastatin, quinidine, disulfiram, ketoconazole and ticlopidine) and CYP-selective inducers (phenobarbital, dexamethasone and ß-naphthoflavone). Residual concentrations of the substrate and metabolites were determined by HPLC, and further identified by their mass spectra and chromatographic behavior. These experiments showed that CYP450 is involved in Pd-Ia metabolism, and that the major CYP isoform responsible is CYP3A1/2, which acts in a concentration-dependent manner. Four Pd-Ia metabolites (M1, M2, M3, and M4) were detected after incubation with rat liver microsomes. Hydroxylation was the primary metabolic pathway of Pd-Ia, and possible chemical structures of the metabolites were identified. Further research is now needed to link the metabolism of Pd-Ia to its drug-drug interactions.

[Systematic analysis of molecular mechanisms of action of omega-3 polyunsaturated fatty acids on arrhythmia].

Safe pharmacotherapy and prevention of arrhythmia is an urgent problem of modern cardiology. Essential micronutrients such as omega-3 polyunsaturated fatty acids (-3 PUF-) significantly contribute to the metabolic processes in cardiomyocytes and cardiac conduction system. This article presents a systematic analysis of anti-arrhythmic effects of omega-3 PUFA and of standardized ethyl esters of omega-3 PUFA. The currently available data indicate two fundamentally different molecular mechanisms of anti-arrhythmic effects: "slow" and "fast" types. Formulation of fundamental prospects of bioinformatic studies of molecular effects of anti-arrhythmic action of omega-3 PUF-s is presented.

Metabolomic study of myocardial ischemia and intervention effects of Compound Danshen Tablets in rats using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Myocardial ischemia (MI) is a worldwide epidemic. Compound Danshen Tablets (CDTs), an herbal compound preparation, are widely used to treat MI in China. In this study, we aimed to explore novel biomarkers to increase the understanding of MI and investigate therapeutic mechanisms of CDT by using a metabolomic approach. Plasma extracts from sham, MI model, CDT- and western medicines (isosorbide dinitrate, verapamil, propranolol, captopril, and trimethazine)-treated rats were analyzed by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The orthogonal partial least square (OPLS) model was built to find metabolites expressed in significantly different amounts between MI and sham rats. Meanwhile, partial least squares discriminant analysis (PLS-DA) was used to investigate CDT's protective effects. The results showed that CDT presented protective effects on MI by reversing potential biomarkers to sham levels, especially for the four metabolites in the pathway of purine metabolism (hypoxanthine, xanthine, inosine and allantoin).

Pleiotropic, cardioprotective effects of omega-3 polyunsaturated fatty acids.

The cardioprotective effects of food rich in omega-3 (omega-3) polyunsaturated fatty acids (PUFA) on cardiovascular risk has been of interest from the moment when a low rate of coronary heart disease was documented in the Eskimo population. The aim of the present review is to discuss recent studies documenting multidirectional action of omega-3 PUFA due to its pleiotropic properties. Experimental studies in cellular and animal models have extensively documented the favorable effects of omega-3 PUFA (eicosapentaenoic acid and docosahexaenoic acid) on: inflammatory processes, endothelial dysfunction, platelet aggregation and arrhythmogenesis. It was reported that antiarrhythmic effects of omega-3 PUFA resulted from stabilization of cardiomyocyte membrane and inhibition of ion channels. Moreover, PUFA possess several pleiotropic properties i.e. anti-inflammatory, anti-atherogenic and antithrombotic. Anti-atherogenic effects (plaque stabilization) of omega-3 PUFA have recently been demonstrated. It was documented (OCEAN study) that eicosapentaenoic acid from a source of highly purified ethyl esters is incorporated into plaques in a relatively short period of time and these higher concentrations of omega-3 PUFA may stabilize vulnerable atherosclerotic plaques. The anti-inflammatory effect of omega-3 PUFA is associated with reduction of levels of TNF-alpha and interleukin-6. Eicosapentaenoic acid and docosahexaenoic acid inhibit arachidonic acid metabolism to inflammatory eicosanoids.

Effects of the selective EET antagonist, 14,15-EEZE, on cardioprotection produced by exogenous or endogenous EETs in the canine heart.

Previously, we demonstrated (17) that 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) produce marked reductions in myocardial infarct size. Although it is assumed that this cardioprotective effect of the EETs is due to a specific interaction with a membrane-bound receptor, no evidence has indicated that novel EET antagonists selectively block the EET actions in dogs. Our goals were to investigate the effects of 11,12- and 14,15-EET, the soluble epoxide hydrolase inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), and the putative selective EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), on infarct size of barbital anesthetized dogs subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. Furthermore, the effect of 14,15-EEZE on the cardioprotective actions of the selective mitochondrial ATP-sensitive potassium channel opener diazoxide was investigated. Both 11,12- and 14,15-EET markedly reduced infarct size [expressed as a percentage of the area at risk (IS/AAR)] from 21.8 +/- 1.6% (vehicle) to 8.7 +/- 2.2 and 9.4 +/- 1.3%, respectively. Similarly, AUDA significantly reduced IS/AAR from 21.8 +/- 1.6 to 14.4 +/- 1.2% (low dose) and 9.4 +/- 1.8% (high dose), respectively. Interestingly, the combination of the low dose of AUDA with 14,15-EET reduced IS/AAR to 5.8 +/- 1.6% (P < 0.05), further than either drug alone. Diazoxide also reduced IS/AAR significantly (10.2 +/- 1.9%). In contrast, 14,15-EEZE had no effect on IS/AAR by itself (21.0 +/- 3.6%), but completely abolished the effect of 11,12-EET (17.8 +/- 1.4%) and 14,15-EET (19.2 +/- 2.4%) and AUDA (19.3 +/- 1.6%), but not that of diazoxide (10.4 +/- 1.4%). These results suggest that activation of the EET pathway, acting on a putative receptor, by exogenous EETs or indirectly by blocking EET metabolism, produced marked cardioprotection, and the combination of these two approaches resulted in a synergistic effect. These data also suggest that 14,15-EEZE is not blocking the mitochondrial ATP-sensitive potassium channel as a mechanism for antagonizing the cardioprotective effects of the EETs.

Biological activity of selected tyrosine-containing 2,5-diketopiperazines.

The study investigates two cyclic dipeptides, cyclo(Tyr-Tyr) (cTT) and cyclo(Phe-Tyr) (cPT) with respect to their biological activity. Investigations using the whole-cell patch-clamp technique testing the effects of 100 microM cyclic dipeptide on ion channels, revealed reversible voltage-dependant blockade for cTT, while cPT exhibited irreversible time-dependant blockade of L-type calcium channels. The isolated retrogradely-perfused rat heart was used to determine the effects of 100 microM of either cTT or cPT on heart rate (HR), coronary flow rate (CFR), left ventricular systolic pressure (LVSP) and cardiac conduction speed. Results indicated opposing effects for the two compounds, where cTT increased HR and CF while cPT decreased HR, CF, LVSP as well as conduction speed. Other biological investigations included opioid binding and anti-neoplastic assays. Competitive binding curves, using tritiated DAMGO, revealed significant binding to micro-opioid receptors with IC50 values for cTT and cPT being 0.82 microM and 69.7 microM respectively. Anti-neoplastic activity was tested using three cultured cell lines: HT-29, MCF-7 and HeLa which were exposed to 2.5 mM cyclic dipeptide or 0.1 mM melphalan as a positive control. While cTT showed little activity against these lines, cPT resulted in as much as a 75.6% growth inhibition of MCF-7 cells, while also being active against HeLa (73.4% inhibition) and HT-29 (60.6%). The results indicate potential biological activity, showing a need for more investigation into tyrosine containing cyclic dipeptides and their analogues as potential bioactive compounds.

Role of metabolically active drugs in the management of ischemic heart disease.

This article reviews the fundamentals of myocardial energy metabolism and selectively outlines the use of several metabolically active drug therapies in the treatment of ischemic heart disease. These drugs - ranolazine, trimetazidine, dichloroacetate (DCA), glucose-insulin-potassium (GIK) solutions, and L-carnitine - have mechanisms of action distinct from traditional anti-ischemic drugs. These agents work by shifting myocardial energy metabolism away from fatty acids toward glucose as a source of fuel. Because these agents are well tolerated and do not affect heart rate or blood pressure, they conceivably could supplement traditional anti-ischemic drug therapy with little risk. The background, rationale for use, and published literature on each agent is reviewed, and the outcomes of pertinent clinical trials are discussed. In the case of ranolazine, data suggest benefit in the treatment of stable angina pectoris, particularly with sustained release formulations. Trimetazidine appears to have similar physiologic effects to ranolazine, and it is effective as monotherapy and as additive therapy in patients with chronic ischemic heart disease. DCA improves acidosis in critically ill patients and, likewise, improves myocardial hemodynamics in those with chronic coronary artery disease and congestive heart failure; however, its metabolism is variable and clinical data on its use in chronic ischemic heart disease are limited. GIK solutions have been shown to be beneficial in animal and human models of ischemia and acute myocardial infarction, and they offer an inexpensive means by which to improve the oxidation of glucose in the heart. Lastly, a large body of literature suggests a benefit with L-carnitine in a number of cardiovascular illnesses, including ischemic heart disease. Clinical trial data in acute myocardial infarction are promising and have prompted the initiation of a large-scale mortality trial.

Hemofiltration: determinants of drug loss and concentration.

Ultrafiltration has proven to be an effective method of reducing post-cardiopulmonary bypass edema and the inflammatory response associated with activation of the complement cascade. Recently, a modified ultrafiltration process has been developed which allows further hemoconcentration after weaning from bypass. Both of these procedures can have an effect upon the concentrations of many of the pharmaceutical agents being used either pre- or perioperatively. Based upon work done by other investigators in the field of dialysis, we have developed sieving coefficients for many cardiovascular drugs in order to provide an estimation of their loss during extended periods of hemofiltration. Our study utilized the pharmacological and physical characteristics for volume of distribution and protein binding to determine the movement of drugs through a hemofilter. Our results demonstrated that concentrations of certain drugs used during open heart surgery may be significantly increased or decreased with the use of hemofiltration techniques.

Beneficial effect of tan-shen, an extract from the root of Salvia, on post-hypoxic recovery of cardiac contractile force.

The present study was undertaken to elucidate the possible effects of tanshinone VI, one of the extracts from the root of Salvia, on post-hypoxic recovery of cardiac contractile force. For this purpose, rat hearts were perfused for 45 min under reoxygenated conditions following 20-min hypoxic perfusion, and changes in tissue high-energy phosphates and calcium contents, and release of ATP metabolites and creatine kinase were examined. Post-hypoxic recovery of cardiac contractile force was augmented when hearts were treated with 42 nM tanshinone VI during hypoxia. This beneficial recovery was accompanied by enhanced restoration of myocardial high-energy phosphates, depression of hypoxia- and reoxygenation-induced increase in tissue calcium content, and suppression of release of ATP metabolites such as adenosine, inosine and hypoxanthine from the perfused heart. The results suggest that tanshinone VI is beneficial for the recovery of cardiac contractility after a certain period of oxygen-deficiency, possibly through mechanisms involving improvement of myocardial energy production upon oxygen-replenishment and/or inhibition of calcium accumulation in the cardiac cell.