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Dietary flaxseed has been shown to have potent antiatherogenic effects in rabbits. The purpose of the present study was to investigate the antiatherogenic capacity of flaxseed in an animal model that more closely represents the human atherosclerotic condition, the LDL receptor-deficient mouse (LDLrKO), and to identify the cellular mechanisms for these effects. LDLrKO mice were administered a regular diet (RG), a 10% flaxseed-supplemented diet (FX), or an atherogenic diet containing 2% cholesterol alone (CH) or supplemented with 10% flaxseed (CF), 5% flaxseed (CF5), 1% flaxseed (CF1), or 5% coconut oil (CS) for 24 wk. LDLrKO mice fed a cholesterol-supplemented diet exhibited a rise in plasma cholesterol without a change in triglycerides and an increase in atherosclerotic plaque formation. The CS mice exhibited elevated levels of plasma cholesterol, triglycerides, and saturated fatty acids and an increase in plaque development. Supplementation of the cholesterol-enriched diet with 10% (wt/wt) ground flaxseed lowered plasma cholesterol and saturated fatty acids, increased plasma ALA, and inhibited plaque formation in the aorta and aortic sinus compared with mice fed a diet supplemented with only dietary cholesterol. The expression of proliferating cell nuclear antigen (PCNA) and the inflammatory markers IL-6, mac-3, and VCAM-1 was increased in aortic tissue from CH and CS mice. This expression was significantly reduced or normalized when flaxseed was included in the diet. Our results demonstrate that dietary flaxseed can inhibit atherosclerosis in the LDLrKO mouse through a reduction of circulating cholesterol levels and, at a cellular level, via antiproliferative and anti-inflammatory actions.

Dietary conjugated linoleic acid (CLA) and the antihypertensive drug, telmisartan, have both been shown to modify cardiovascular risks. The effects of a combination of these two agents have, however, not been investigated. This 20 week study sought to assess the therapeutic potential of a CLA/telmisartan co-administration in rats fed a high-fructose high-fat diet. Thirty-three male Sprague-Dawley rats were randomly assigned to five experimental groups, including control, losartan, telmisartan, CLA, and CLA + telmisartan-treated animals. Body weight, blood pressure, and blood levels of lipids, glucose, insulin, and inflammatory markers were measured. Co-administration of CLA and telmisartan resulted in significant (P0.05) reductions in body weight, visceral fat, serum total cholesterol, triglycerides, glucose, plasma insulin concentrations, and systolic blood pressure compared with those in the control group. Moreover, plasma levels of IL1-alpha and IFN-gamma were reduced and levels of IL1-beta, IL-4, IL-6, and IL-10, plus TNF-alpha were increased in the co-therapy group, compared with controls. In conclusion, this study suggests that a combination of CLA with telmisartan may modify several risk factors of cardiovascular disease commonly seen in metabolic syndrome. This combination of nutraceuticals and pharmaceuticals may be a safe and cost-effective strategy in a number of high-risk subjects. Future studies will further document clinical benefits of such combination therapy.

Although it is known that mechanical stretching of cells can induce significant increases in cell growth and shape, the intracellular signaling pathways that induce this response at the level of the cell nucleus is unknown. The transport of molecules from the cell cytoplasm to the nucleoplasm through the nuclear pore is a key pathway through which gene expression can be controlled in some conditions. It is presently unknown if mechanical stimuli can induce changes in nuclear pore expression and/or function. The purpose of the present investigation was to determine if mechanical stretching of a cell will alter nuclear protein import and the mechanisms that may be responsible. Vascular smooth muscle cells that were mechanically stretched exhibited an increase in proliferating cell nuclear antigen expression, cell number, and cell size within 24-48 h. Cells were microinjected with marker proteins for nuclear import. Nuclear protein import was significantly stimulated in stretched cells when compared with control. This was associated with an increase in the expression of nuclear pore proteins as detected by Western blots. Inhibition of the MAPK pathway blocked the stretch-induced stimulation of both cell proliferation and nuclear protein import. We conclude that nuclear protein import and nuclear pore density can adapt to mechanical stimuli during the process of cell growth through a MAPK-mediated mechanism.

This study was conducted to investigate the rutin content of three buckwheat species: Fagopyrum esculentum, Fagopyrum tataricum and Fagopyrum homotropicum, and to evaluate their antioxidant capacity. In total, 11 cultivars/accessions were analyzed. The contents of both rutin and total flavonoids were significantly different depending on species, 0.02% and 0.04% in F. esculentum, 0.10% and 0.35% in F. homotropicum, and 1.67% and 2.04% in F. tataricum, respectively. Three buckwheat species exhibited a dose–response effect in inhibiting low-density lipoprotein (LDL) peroxidation. The antioxidant activity decreased in the order: F. tataricum > F. homotropicum > F. esculentum. Linear regression analysis revealed a correlation between antioxidant activity and rutin content (R2 = 0.98) or total flavonoids content (R2 = 0.77) in all buckwheat cultivars/accessions. This work shows that rutin plays an important role in antioxidant activity of buckwheat seed. It provides useful information for buckwheat breeding to develop high rutin content varieties.

Dietary intake of omega-3 polyunsaturated fatty acids (PUFA) like alpha-linolenic acid (ALA) is antiarrhythmic and cardioprotective. PUFA may also be beneficial in hypertension. Altered Na(+)-Ca(2+) exchanger (NCX) activity has been implicated in arrhythmias, hypertension and heart failure and may be a target for PUFA. Thus, we tested the effects of ALA and other distinct fatty acids on the cardiac (NCX1.1) and vascular (NCX1.3) NCX isoforms.HEK293 cells stably expressing NCX isoforms were ramped from +60 to -100 mV (over 1600 ms) in the absence and presence of 25 microM oleic acid (OA, omega-9), linoleic acid (LA, omega-6), ALA (omega-3), or eicosapentaenoic acid (EPA, omega-3). NiCl(2) (5 mM) was used to inhibit and therefore identify the NCX current. The effect of 25 microM ALA on NCX1.1 and NCX1.3 activity was also assessed in adult rat ventricular cardiomyocytes and rabbit aortic vascular smooth muscle cells (VSMC) by measuring [Ca(2+)](i) following substitution of [Na(+)](o) with Li(+).Application of Ni(2+) had no effect in non-transfected cells. ALA and EPA (25 microM) reduced the Ni(2+)-sensitive forward NCX1.1 current (at -100 mV) by 64% and reverse current (at +60 mV) by 57%, and inhibited the Ni(2+)-sensitive NCX1.3 forward and reverse currents by 79% and 76%, respectively. Neither OA nor LA (25 microM) affected the NCX1.1 currents, but both partially inhibited the forward and reverse mode NCX1.3 currents. Inhibition of NCX1.3 by ALA occurred at a much lower IC(50) ( approximately 19 nM) than for NCX1.1 ( approximately 120 nM). In cardiomyocytes and VSMC, ALA significantly reduced the Li(+)-induced rise in intracellular [Ca(2+)].NCX1.3 is more sensitive to inhibition by ALA than NCX1.1. In addition, only omega-3 PUFA inhibits NCX1.1, but several classes of fatty acids inhibit NCX1.3. The differential sensitivity of NCX isoforms to fatty acids may have important implications as therapeutic approaches for hypertension, heart failure and arrhythmias.

Dietary trans-fats are strongly associated with heart disease. However, the capacity for the tissues of the body, and specifically the heart, to take up trans-fats is unknown. It is also unknown if different trans-fats have different uptake capacities in the heart and other tissues of the body. Diets of low-density lipoprotein receptor-deficient mice were supplemented for 14weeks with foods that contained 1.5% of the trans-fat elaidic acid or vaccenic acid. Tissues were extracted and frozen in liquid nitrogen, and then lipids were analyzed by gas chromatography for fatty acid content. Isolated cardiomyocytes were also exposed to elaidic or vaccenic acid in cell culture media for 24h. Dietary supplementation with vaccenic or elaidic acid resulted in a 20-fold higher accumulation of these TFAs in fat deposits in the body in comparison to liver. Liver tissue accumulated about twice as much per gram tissue as heart. Similar quantities of both elaidic acid and vaccenic acid were taken up by the tissues. Isolated cardiomyocytes exhibited an unusually large uptake of trans-fat, and this was dependent upon both the concentration and duration of exposure to the trans-fats but not upon the type of trans-fat. Expression levels of CD36 and FATP4 were not significantly changed during dietary interventions or exposure of cells to trans-fats. We conclude that fat, liver and heart (including cardiomyocytes) are all capable of accumulating trans-fat in response to dietary supplementation without changes in fatty acid transport protein expression.

ERK-2 MAP kinase activation induces inhibitory effects on nuclear protein import in vascular smooth muscle cells. The mechanism and characteristics of this effect of ERK-2 were investigated. An unusual dose-dependent effect of ERK-2 on nuclear protein import was identified. At higher concentrations (1 μg/mL) of ERK-2, nuclear protein import was stimulated, whereas lower concentrations (0.04 μg/mL) inhibited import. Intermediate concentrations exerted intermediate effects. The stimulatory and inhibitory effects at the 2 different ERK-2 concentrations were observed in both conventional, permeabilized cell assays of nuclear protein import and with in situ microinjection of smooth muscle cells. The biphasic effects of ERK-2 on import were also found for the other 2 members of the MAPK family, p38 and JNK. RanGAP was identified by structural analysis as a candidate target protein responsible for mediating the effects of ERK-2. After pretreatment with high concentrations of ERK-2, RanGAP activity was significantly increased by ~50%. In contrast, low concentrations of ERK-2 significantly attenuated RanGAP activity. These results demonstrate that all 3 members of the MAPK family can alter nuclear protein import in opposite directions depending upon the concentration of ERK-2 used. RanGAP represents the MAP kinase target whereby nuclear transport can be stimulated or inhibited.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disorders. Elevated plasma homocysteine (Hcy) concentration is associated with other cardiovascular risk factors. We previously reported that Hcy stimulated cholesterol biosynthesis in HepG2 cells. In the present study, we investigated the underlying mechanisms of Hcy-induced hepatic cholesterol biosynthesis in an animal model. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 wk. The mRNA expression and the enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were significantly increased in livers of hyperhomocysteinemic rats. There were marked hepatic lipid accumulation and an elevation of plasma cholesterol concentration in hyperhomocysteinemic rats. Three transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element-binding protein (CREB), and nuclear factor Y (NF-Y) were activated in livers of hyperhomocysteinemic rats. Upon Hcy treatment of hepatocytes, there was a significant increase in HMG-CoA reductase mRNA expression in these cells. The activation of SREBP-2, CREB, and NF-Y preceded the increase in HMG-CoA reductase expression in Hcy-treated cells. Pretreatment of hepatocytes with inhibitors for transcription factors not only blocked the activation of SREBP-2, CREB, and NF-Y but also attenuated Hcy-induced HMG-CoA reductase mRNA expression. These results suggested that hyperhomocysteinemia-induced activation of SREBP-2, CREB, and NF-Y was responsible for increased cholesterol biosynthesis by transcriptionally regulating HMG-CoA reductase expression in the liver leading to hepatic lipid accumulation and subsequently hypercholesterolemia. In conclusion, the stimulatory effect of Hcy on hepatic cholesterol biosynthesis may represent an important mechanism for hepatic lipid accumulation and cardiovascular disorder associated with hyperhomocysteinemia.

The cardiac Na + –Ca 2+ exchanger (NCX1) is the main mechanism for Ca 2+ efflux in the heart and is thought to serve an essential role in cardiac excitation-contraction (E-C) coupling. The demonstration that an NCX1 gene knock-out is embryonic lethal provides further support for this essential role. However, a recent report employing the Cre/loxP technique for cardiac specific knock-out of NCX1 has revealed that cardiac function is remarkably preserved in these mice, which survived to adulthood. This controversy highlights the necessity for further investigation of NCX1 function in the heart. In this study, we report on a novel approach for depletion of NCX1 in postnatal rat myocytes that utilizes RNA interference (RNAi), administered with high efficiency via adenoviral transfection. Depletion of NCX1 was confirmed by immunocytochemical detection, Western blots and radioisotopic assays of Na + –Ca 2+ exchange activity. Exchanger expression was inhibited by up to ~94%. Surprisingly, spontaneous beating of these cardiomyocytes was still maintained, although at a lower frequency. Electrical stimulation could elicit a normal beating rhythm, although NCX depleted cells exhibited a depressed Ca 2+ transient amplitude, a depressed rate of Ca 2+ rise and decline, elevated diastolic [Ca 2+ ], and shorter action potentials. We also observed a compensatory increase in sarcolemmal Ca 2+ pump expression. Our data support an important, though non-essential, role for the NCX1 in E-C coupling in these neonatal heart cells. Furthermore, this approach provides a valuable means for assessing the role of NCX1 and could be utilized to examine other cardiac proteins in physiological and pathological studies.

Nutraceuticals may induce doping infractions through contamination of the product itself or their ingestion might be metabolized within the body to create a positive doping control test. We tested this possibility using a commercially available, proprietary ginseng root extract (Cold-FX®, CV Technologies Inc., Edmonton, AB). After athletes ingested Cold-FX© for 28 d at 400 mg/d, urine samples were collected and processed under strict IOC doping control guidelines and then analyzed for a full screen of IOC banned/restricted substances by an IOC-approved laboratory. There were no positive tests for any banned substances in any of the subjects. Our study demonstrates that ingestion of Cold-FX® for 28 d at 400 mg/d does not represent a doping concern for athletes. Carefully controlled clinical studies like this one are necessary to provide the athlete, the nutraceutical industry and IOC regulatory bodies with information to avoid inadvertent exposure to banned/restricted or potentially unhealthy substances.

Red wine and its components have been shown to possess cardioprotective and anti-atherogenic effects. Additionally, red wine and many of its components like catechin, epicatechin, rutin, transresveratrol and quercetin possess antioxidant properties. Oxidized low density lipoprotein (LDL) is involved in the development of an atherosclerotic lesion. Red wine, therefore, may be anti-atherogenic because of its antioxidant effects on LDL modification. This study examined the antioxidant effects of catechin, epicatechin, rutin, transresveratrol, quercetin and Merlot wines on LDL oxidation. Merlot was chosen because although other red wines have been tested, limited information exists for this variety. Oxidation was carried out with AAPH (2,2'-Azo-bis(2-amidinopropane) dihydrochloride) and AMVN (2,2'-Azo-bis(2,4-dimethylvaleronitrile)), as water and lipid soluble peroxyl radical generating systems (FRGS), respectively. This allowed us to determine the lipophilic antioxidant characteristics of the wine and its components. Conjugated diene assays were used to measure LDL oxidation over 6 hrs. In an AAPH system, all polyphenolic compounds except transresveratrol displayed an antioxidant effect. LDL oxidation by AAPH was also inhibited by aliquots of Merlot wine. No antioxidant effects were observed in an AMVN environment except for a mild antioxidant effect by quercetin. Surprisingly, incubation of LDL with Merlot wine strongly protected against oxidation by AMVN. In summary, the five phenolic compounds displayed antioxidant effects in a water soluble free radical generating system, but only quercetin showed this in a lipid soluble one. However, red wine inhibited LDL oxidation by both water and lipid soluble free radical generating systems. Our data suggest, therefore, that red wines contain unidentified antioxidants that provide protection against LDL oxidation within a lipid soluble environment. (Mol Cell Biochem 263: 211-215, 2004).

We have previously shown that infection with Chlamydia pneumoniae can significantly exacerbate atherosclerotic lesions in LDLR-/- mice concurrently fed a high cholesterol diet in 6 or 9 months. We now report that a period of 4 month was sufficient for demonstrating the C. pneumoniae atherogenicity. However, heat inactivation of C. pneumoniae organisms completely abolished the ability of C. pneumoniae to exacerbate the atherosclerotic lesions, suggesting that viable organism infection may be required for the C. pneumoniae atherogenicity.

Objective— Our study tested the hypothesis that the mitogenic effect of oxidized low-density lipoprotein (oxLDL) on vascular cells may be further enhanced by the presence of cytokines and growth factors known to be present in the atherosclerotic environment. Methods and Results— Quiescent fibroblasts and vascular smooth muscle cells were treated with 10 or 50 μg/mL minimally-oxidized LDL in combination with serum for 24 or 48 hours. Surprisingly, these cells showed inhibited release from growth arrest and a significant reduction in the number of cells completing the cell cycle when compared with cells treated with serum alone. This was not due to an induction of apoptosis. The antiproliferative effects were not closely associated with changes in the expression of cell cycle proteins. Instead, oxLDL inhibited the translocation of cell cycle proteins cell division cycle (Cdc) 2, cyclin-dependent kinase (Cdk) 2, Cdk 4, Cyclin A, Cyclin B1, Cyclin D1, and proliferative cell nuclear antigen (PCNA) into the nucleus, as compared with separate treatments with serum alone. Kinase activation associated with specific cell cycle proteins was also inhibited by oxLDL. Conclusions— oxLDL, in the presence of serum, has a surprising inhibitory effect on cell proliferation that occurs through an inhibition of import of cell cycle proteins into the cell nucleus.

The effects of exogenous phosphatidic acid (PA) on Ca2+ transients and contractile activity were studied in cardiomyocytes isolated from chronic streptozotocin-induced diabetic rats. In control cells, 25 μM PA induced a significant increase in active cell shortening and Ca2+ transients. PA increased IP3 generation in the control cardiomyocytes and its inotropic effects were blocked by a phospholipase C inhibitor. In cardiomyocytes from diabetic rats, PA induced a 25% decrease in active cell shortening and no significant effect on Ca2+ transients. Basal and PA-induced IP3 generation in diabetic rat cardiomyocytes was 3-fold lower as compared to control cells. Sarcolemmal membrane PLC activity was impaired. Insulin treatment of the diabetic animals resulted in a partial recovery of PA responses. Our results, therefore, identify an important defect in the PA-PLC signaling pathway in diabetic rat cardiomyocytes, which may have significant implications for heart dysfunction during diabetes.

Genome analysis has revealed the presence of key components of the Na+ chemiosmotic cycle, including the primary Na+ pump (Na+-translocating NADH:ubiquinone oxidoreductase), in the cytoplasmic membrane of two ubiquitous human pathogens, Chlamydia trachomatis and Chlamydiophyla pneumoniae. This observation seemed paradoxical in the case of obligatory intracellular parasites because the Na+ cycle is thought to be primarily a mechanism that enhances the adaptive potential in free-living bacteria that are often facing drastic changes in the salinity and pH of the environment. We present a model suggesting that operation of the Na+ cycle may play an important role in the course of chlamydial infection, when the Na+ and H+ homeostasis of the host cell become severely impaired. This introduces the intriguing possibility of the application of drugs targeting Na+-transporting enzymes to chlamydial infections, which are notoriously difficult to treat.

Chlamydia pneumoniaeinfection has been linked with atherosclerosis. However, the mechanism responsible for the atherogenic effects ofC pneumoniaeremains unclear. Heat shock proteins (HSPs) have been found in atherosclerotic lesions. HSPs of HSP70 and HSP90 families are involved in the regulation of cell cycle progression and cell proliferation. We assessed the hypothesis that HSP60 is induced in vascular cells infected withC pneumoniaeand stimulates cell proliferation. Rabbit vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were infected withC pneumoniae. Western blot analysis demonstrated the induction of endogenous HSP60 expression inC pneumoniae-infected VSMCs.C pneumoniaeinfection significantly increased the number of VSMCs, and the mitogenic effect correlated with the expression level of endogenous HSP60. In contrast to VSMCs,C pneumoniaeinfection had no effect on the expression level of HSP60 and did not stimulate cell proliferation in HUVECs. Exogenous addition of recombinant chlamydial HSP60 had no mitogenic effect on VSMCs and HUVECs. However, overexpression of HSP60 within VSMCs by infection with adenovirus encoding human HSP60 resulted in a significant increase in cell numbers compared with uninfected VSMCs. These results suggest that overexpression of endogenous HSP60 may be a central intracellular event responsible for the mitogenic effects induced byC pneumoniaeinfection. In addition toC pneumoniae, other infectious agents and atherogenic risk factors may also stimulate VSMC proliferation and contribute to the lesion formation through the induction of HSP60.

Myocardial ischemia-reperfusion activates the Na+/H+exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The ω-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na+/H+exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na+/H+exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10–100 μM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H+-dependent22Na+uptake was inhibited by 30–50% after treatment with ≥50 μM EPA or ≥25 μM DHA. This was a specific effect of these PUFAs, because 50 μM linoleic acid or linolenic acid had no significant effect on Na+/H+exchange. The SL vesicles did not exhibit an increase in passive Na+efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na+/H+exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.

Heat shock proteins play important housekeeping roles in a variety of cells within the body during normal control conditions. The many different functions for heat shock proteins in the cell depend upon the specific heat shock protein involved. Each protein is nominally differentiated based upon its molecular size. However, in addition to their role in normal cell function, heat shock proteins may play an even more important role as pro-survival proteins conserved through evolution to protect the cell from a variety of stresses. The ability of a cell to withstand these environmental stresses is critical to its capacity to adapt and remain viable. Loss of this ability may lead to pathological states. Abnormal localization, structure or function of the heat shock proteins has been associated with many pathologies, including those involving heart disease. Heat shock proteins like HSP60 and HSP70 in particular have been identified as playing important roles in inflammation and immune reactions. Inflammation has been identified recently as an important pathological risk factor for heart disease. It is perhaps not surprising therefore, that heat shock protein family has been increasingly identified as an important intracellular pathway associated with inflammatory-mediated heart conditions including atherosclerosis. This paper reviews the evidence in support of a role for heat shock proteins in cardiovascular disease and the potential to target these proteins to alter the progression of atherosclerotic disease.

Aims During cardiac hypertrophy, cardiomyocytes (CMs) increase in the size and expression of cytoskeletal proteins while reactivating a foetal gene programme. The process is proposed to be dependent on increased nuclear export and, since nuclear pore trafficking has limited capacity, a linked decrease in import. Our objective was to investigate the role of nuclear import and export in control of hypertrophy in rat and human heart failure (HF). Methods and results In myocardial tissue and isolated CMs from patients with dilated cardiomyopathy, nuclear size was increased; Nucleoporin p62, cytoplasmic RanBP1, and nuclear translocation of importins (α and β) were decreased while Exportin-1 was increased. CM from a rat HF model 16 weeks after myocardial infarction (MI) reproduced these nuclear changes. Nuclear import, determined by the rate of uptake of nuclear localization sequence (NLS)-tagged fluorescent substrate, was also decreased and this change was observed from 4 weeks after MI, before HF has developed. Treatment of isolated rat CMs with phenylephrine (PE) for 48 h produced similar cell and nuclear size increases, nuclear import and export protein rearrangement, and NLS substrate uptake decrease through p38 MAPK and HDAC-dependent pathways. The change in NLS substrate uptake occurred within 15 min of PE exposure. Inhibition of nuclear export with leptomycin B reversed established nuclear changes in PE-treated rat CMs and decreased NLS substrate uptake and cell/nuclear size in human CMs. Conclusions Nuclear transport changes related to increased export and decreased import are an early event in hypertrophic development. Hypertrophy can be prevented, or even reversed, by targeting import/export, which may open new therapeutic opportunities.

Background In 2013 the World Health Organization deemed hypertension as a global crisis as it is the leading risk factor attributed to global mortality. Therefore, there is a great need for effective alternative treatment strategies to combat a condition that affects 40% of adults worldwide. Recently, the FlaxPAD Trial observed a significant reduction in systolic and diastolic blood pressure in hypertensive patients with peripheral arterial disease that consumed 30 g of milled flaxseed per day for one year. However, these patients were already on anti-hypertensive medication. Therefore, there is a need to assess if dietary flaxseed can effectively reduce blood pressure in the absence of peripheral arterial disease and anti-hypertensive medication in newly diagnosed hypertensive patients. Methods/Design The HYPERFlax Trial is a parallel, superiority, phase II/III, randomized, double-blinded, controlled clinical trial. St. Boniface Hospital and the Health Sciences Centre of Winnipeg, Canada, will recruit 100 participants newly diagnosed with stage 1 hypertension who have yet to be administered anti-hypertensive medication. Participants will be randomly allocated with a 1:1 ratio into a flaxseed or control group and provided food products to consume daily for six months. At baseline, two, four, and six months, participant assessments will include the primary outcome measure, averaged automated blood pressure, and secondary measures: 24-hour food recall, international physical activity questionnaire, anthropometrics, and blood and urine sampling for biochemical analysis. Plasma will be assessed for lipids, metabolomics profiling, and molecules that regulate vascular tone. Urine will be collected for metabolomics profiling. With an estimated dropout rate of 20%, the trial will have a power of 0.80 to detect differences between groups and across time, out of an effect size of 0.7 (SD) at an α level of 0.05. Discussion This trial will determine if dietary flaxseed is efficacious over six months as an anti-hypertensive therapy in subjects newly diagnosed with hypertension. If flaxseed can effectively reduce blood pressure as a monotherapy, then flaxseed will provide individuals on a global basis with a cost-effective food-based strategy to control hypertension. Trial registration NCT01952340, Registered 24 September 2013.

Oxylipins, including eicosanoids, are highly bioactive molecules endogenously produced from polyunsaturated fatty acids. Oxylipins play a key role in chronic disease progression. It is possible, but unknown, if oxylipin concentrations change with the consumption of functional foods or differ with subject age.Therefore, in a parallel comparator trial, 20 healthy individuals were recruited into a younger (19-28years) or older (45-64years) age group (n=10/group). Participants ingested one muffin/day containing 30g of milled flaxseed (6g alpha-linolenic acid) for 4weeks. Plasma oxylipins were isolated through solid phase extraction, analyzed with HPLC-MS/MS targeted lipidomics, and quantified with the stable isotope dilution method.At baseline, the older group exhibited 13 oxylipins ≥2-fold the concentration of the younger group. Specifically, pro-inflammatory oxylipins 5-hydroxyeicosatetraenoic acid, 9,10,13-trihydroxyoctadecenoic acid, and 9,12,13-trihydroxyoctadecenoic acid were significantly greater in the older (1.1±0.23nM, 5.6±0.84nM, and 4.5±0.58nM, respectively) versus the younger group (0.34±0.12nM, 3.5±0.33nM, and 3.0±0.24nM, respectively) (p0.05). After 4weeks of flaxseed consumption the number of oxylipins that were ≥2-fold higher in the older versus the younger group was reduced to 3. 5-Hydroxyeicosatetraenoic acid, 9,10,13-trihydroxyoctadecenoic acid, and 9,12,13-trihydroxyoctadecenoic acid decreased in the older group to concentrations equivalent to the younger group after flaxseed consumption.These data suggest a potential role for oxylipins in the aging process and how nutritional interventions like flaxseed can beneficially disrupt these biological changes associated with inflammation and aging.

Cardiovascular disease remains a leading cause of mortality worldwide. It is now clear that myriad modifiable risk factors cause the majority of serious and chronic cardiovascular diseases. Most of these risk factors can be modified through changes in lifestyle and/or with pharmacotherapy. Advances in drug therapy for cardiovascular disease have had a large impact on heart disease over the past six decades. Currently, however, scientific investigations have expanded their focus to study the potential for foods and nutrients that provide the body with health benefits beyond normal nutrition. Many of these ‘functional foods’ or ‘nutraceuticals’ have demonstrated direct benefit for cardiovascular health – for both primary and secondary prevention of cardiovascular disease. Flaxseed is an example of a functional food that has demonstrated significant cardioprotective effects such as lowering the risk of sudden cardiac death or recurrent myocardial infarction. It has demonstrated antiarrhythmic, antihypertensive, antioxidant and lipid-lowering properties. Many of these beneficial effects have been attributed to the rich content of alpha-linolenic acid found in flaxseed. The effects of alpha-linolenic acid on cardiovascular disease, either delivered as a nutraceutical extract or through ingestion of flaxseed in the diet, are discussed in detail.

Sodium-hydrogen exchange is involved in ischemia-reperfusion injury. Despite the strong research support for the use of sodium-hydrogen exchange blockers to protect the myocardium during ischemia-reperfusion, there is still controversy regarding the most effective time for drug delivery. Studies that initiate drug treatment immediately before or at reperfusion have given conflicting results. The conflict in results may be due to differences in the experimental design. Post-ischemic cardioprotection may be less than optimal due to the vascular permeability barrier or a limited collateral circulation. Increasing the drug dose or allowing more time for the drug to cross the vessel wall may overcome this limitation. Determining the correct dose and delivery protocol, therefore, will be critical for the generation of positive results in future clinical trials and will optimize the beneficial effects of sodium-hydrogen exchange inhibition in the treatment of reperfusion injury.

Stimulation of Na(+)/H(+)exchange during ischemia-reperfusion results in cardiac damage. However, it is unclear whether the Na(+)/H(+)exchanger is active during the ischemic period or during reperfusion. Adult beating cardiomyocytes were exposed to an ischemia mimetic solution for 90 min and then reperfused with a normal solution for 30 min. 5-(N,N-dimethyl)-amiloride (DMA), a blocker of the Na(+)/H(+)exchanger, was administered during ischemia and the first 3 min of reperfusion or only during the first 3 min of reperfusion. Administration of DMA only upon reperfusion resulted in increased cell survival (81+/-1%, P

Nuclear protein import plays a critical role in both proliferation and apoptosis. Both actions are of great importance in vascular development and pathology. Consequently, a technique that would allow us to characterize import and identify novel cofactors important in modulating the process in smooth muscle cells is of great applicability. In this article, we describe a simple and reliable nuclear protein import assay that we have modified for use on aortic smooth muscle cells in culture. Briefly, the procedure permeabilizes the cells and analyzes the nuclear retention of a fluorescent import marker. Using this method, we are able to analyze the effect of agents on nuclear protein import. Most importantly, we are able to treat the cytosol, nucleus or the whole cell independently. This technique will allow for the identification and development of drugs to inhibit or stimulate the process and, potentially, to identify mechanistic insight into disease processes.

Cardiac sarcolemmal Na+/H+exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na+/H+exchange. The present study was carried out to determine the effects on Na+/H+exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 ± 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 ± 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 ± 2% inhibition of Na+/H+exchange. Na+/H+exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na+. All of these parameters of Na+/H+exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na+was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na+/H+exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na+/H+exchange.

Diabetes is a serious medical and financial burden on western societies. It is the seventh leading cause of death in the United States and Canada. The disease is due to a primary defect in glucose tolerance and carbohydrate metabolism resulting from either a deficiency of insulin (Insulin-dependent (type I) diabetes mellitus - IDDM) or a state of insulin resistance (Non-insulin-dependent (type II) diabetes mellitus - NIDDM). NIDDM comprises greater than 80% of total diabetic cases. Associated with the primary metabolic defects are equally deleterious secondary complications affecting the renal, ocular, nervous and cardiovascular systems. The cardiovascular complications account for a major proportion of diabetic mortality. As such, it is of paramount importance to develop or find an animal model expressing complications homologous to the human condition. Many models of NIDDM are available to the diabetic researcher but choosing an accurate one can be difficult. The following compares the advantages and limitations of one such model, the JCR:LA-cp rat to other NIDDM models commonly used today.

Abstract —The purpose of the present investigation was to investigate the subcellular basis responsible for the loss of vasoreactivity in atherosclerotic vessels. We have chosen to focus on the potential of oxidized low density lipoprotein (oxLDL), an important atherogenic agent, to alter sarcoplasmic reticulum (SR) structure and function. Vascular smooth muscle cells (VSMCs) were exposed for 1 to 6 days to low concentrations of minimally oxidized LDL. ATP was used to probe SR function in VSMCs. ATP can increase [Ca 2+ ] i in control VSMCs because of a release of Ca 2+ from the SR. However, after chronic exposure to oxLDL, cells lose their ability to increase [Ca 2+ ] i in response to ATP. These cells also exhibit a depressed rise in [Ca 2+ ] i after exposure to ryanodine. These effects were associated with a decreased immunoreactivity for the ryanodine-sensitive Ca 2+ -release channels in the SR of oxLDL-treated cells. Immunohistochemical analysis of aortic sections obtained from rabbits fed a cholesterol-supplemented diet revealed a significant decrease in the immunoreactivity for ryanodine channels in the plaque and in the medial layer underlying the plaque. In summary, our data identify oxLDL as a component within the atherosclerotic milieu capable of inducing a decrease in smooth muscle ryanodine channel density. This alteration is associated with a significant defect in the ability of the SR within the smooth muscle cell to regulate Ca 2+ . These lesions may contribute to the altered vasoreactivity exhibited by atherosclerotic vessels.

H(2)O(2) alters gene expression in many cell types. Alterations in nuclear import of transcription factors or similar key proteins may be responsible for these changes. To investigate this possibility, a cytosolic nuclear import cocktail was treated with varying ¿H(2)O(2) and used in import assays. H(2)O(2) caused a dose- and time-dependent inhibition of import at concentrations as low as 100 microM. Catalase reversed this effect. H(2)O(2) treatment of permeablized cells did not affect import, suggesting that H(2)O(2) was acting on a cytosolic factor. Treatment of import cocktail with two different free radical generating systems had no effect, but treatment of permeablized cells inhibited import, suggesting H(2)O(2) works via a distinct process from hydroxyl or superoxide radicals. Pretreatment of import cocktail with genistein reversed the effect of H(2)O(2) on import. Western blotting revealed that H(2)O(2) activated ERK2. The specific MEK1/2 inhibitor, PD98059, completely blocked the effects of H(2)O(2) on import. Activated ERK2 mimicked H(2)O(2)'s effect on import. Immunocytochemistry revealed that H(2)O(2) treatment of whole cells increased cytosolic Ran/TC4 levels, an effect reversible by catalase or PD98059. These data demonstrate that H(2)O(2) inhibits nuclear protein import and that this effect is mediated by mitogen-activated protein (MAP) kinase activation, possibly by altering Ran/TC4 function.

Oxidized low density lipoprotein (oxLDL) has been identified as a potentially important atherogenic factor. Atherosclerosis is characterized by the accumulation of lipid and calcium in the vascular wall. OxLDL plays a significant role in altering calcium homeostasis within different cell types. In our previous study, chronic treatment of vascular smooth muscle cells (VSMC) with oxLDL depressed Ca2+i homeostasis and altered two Ca2+ release mechanisms in these cells (IP3 and ryanodine sensitive channels). The purpose of the present study was to further define the effects of chronic treatment with oxLDL on the smooth muscle sarcoplasmic reticulum (SR) Ca2+ pump. One of the primary Ca2+ uptake mechanisms in VSMC is through the SERCA2 ATPase calcium pump in the sarcoplasmic reticulum. VSMC were chronically treated with 0.005-0.1 mg/ml oxLDL for up to 6 days in culture. Cells treated with oxLDL showed a significant increase in the total SERCA2 ATPase content. These changes were observed on both Western blot and immunocytochemical analysis. This increase in SERCA2 ATPase is in striking contrast to a significant decrease in the density of IP3 and ryanodine receptors in VSMC as the result of chronic treatment with oxLDL. This response may suggest a specific adaptive mechanism that the pump undergoes to attempt to maintain Ca2+ homeostasis in VSMC chronically exposed to atherogenic oxLDL.

We have previously shown that infection with the C. pneumoniae AR39 strain once monthly for 9 consecutive months significantly exacerbated atherosclerosis in mice with LDL receptor deficiency (LDLR–/–) in the presence of a high cholesterol diet. To further optimize the LDLR–/– mouse model for studying the mechanisms of C. pneumoniae atherogenesis, we have tested a different infection protocol with intranasal inoculation twice monthly for 6 consecutive months in the present study. We found that C. pneumoniae infection for 6 months was sufficient to produce a 130%, significantly greater exacerbation of aortic atherosclerosis in LDLR–/– mice in the presence of a high cholesterol diet. Mice receiving a high cholesterol diet alone displayed a lesion area index of 18.2 ± 6.1 (S.D.) while mice treated with both the high cholesterol diet and C. pneumoniae infection had a lesion area index of 41.8 ± 15.2 (S.D.). However, the chlamydial infection did not significantly alter the mouse serum total cholesterol or the LDL levels induced by the high cholesterol diet. This study not only confirms our previous findings that C. pneumoniae infection can exacerbate aortic atherosclerosis lesion in the LDLR–/– mice, but also further optimizes the LDLR–/– mouse model for future mechanism studies.

Background: Although domoic acid (DA), a shellfish neurotoxin, carries a negative surface charge at physiological pH like that of adenosine-5'-triphosphate (ATP), very little is known about its cellular effects. In view of the potentially significant role of extracellular ATP as a signaling molecule for increasing the intracellular concentration of Ca2+ ([Ca2+]i), we examined the possibility that DA may interfere with this signal transduction mechanism in the myocardium. Methods and Results: Cardiomyocytes were isolated from rat heart and loaded with Fura-2 to measure the [Ca2+]i. ATP produced a gradual rise in [Ca2+]i, reaching a peak level in 25- 30 seconds and declining thereafter. DA did not affect the [Ca2+]i in cardiomyocytes; however, it diminished the ATP-induced elevation in [Ca2+]i in a concentration-dependent manner. Kainic acid, an analogue of DA, had a similar effect but at a 25-fold higher concentration, whereas glutamate and aspartate did not modify the action of ATP. Well-known inhibitors of L-type voltage-sensitive Ca2+ channels, nifedipine and nicardipine, depressed the ATP- induced increase in [Ca2+ ]i, but DA did not produce additive effects with either of these agents. On the other hand, DA potentiated the KCl-induced increase in [Ca2+]i in quiescent cardiomyocytes and augmented the nicardipine-sensitive Ca2+ transients in electrically stimulated cardiomyocytes. Conclusions: These results suggest that DA may diminish the ATP-induced increase in [Ca 2+]i by inhibiting the ATP interaction with cardiomyocytes in a specific manner.

The present study was undertaken to comprehensively characterize low-flow ischemia and reperfusion in single adult cardiomyocytes and to determine whether it is important to control contractile activity. The ischemia-mimetic solution was hypoxic, acidic (pH 6.0), and deficient in glucose but contained elevated KCl. Cardiomyocytes were stimulated to contract throughout ischemia and during reperfusion with control perfusate. After the ischemia-reperfusion insult, cells exhibited poor recovery of active cell shortening, a decrease in passive cell length, increased frequency of necrosis, lower ATP content, and evidence of the generation of oxygen-derived free radicals within the cells. Intracellular lactate concentration increased, pH decreased, and Ca2+transients were depressed during the ischemic insult, but the latter two parameters recovered partially on reperfusion. Basal intracellular Ca2+concentration was elevated during ischemia and early into reperfusion. Recovery was attenuated in cells that were electrically stimulated to contract throughout ischemia. The duration of ischemia, stimulation frequency, and composition of the ischemia-mimetic solution were important variables. The inclusion of 10 mM lactate in the ischemia-mimetic solution significantly aggravated all the parameters examined above. Our data demonstrate that 1) an ischemia-mimetic solution administered to single, isolated adult cardiomyocytes can reproduce many of the responses observed in whole hearts, 2) caution should be used in adding lactate to an ischemic solution, and 3) it is important to stimulate contractile activity throughout ischemia to reproduce the effects of ischemia in whole hearts.

Depressed myofibrillar Ca2+-ATPase activity and sarcoplasmic reticulum (SR) Ca2+uptake are important mechanisms that are responsible for the cardiac dysfunction exhibited by insulin-deficient (type I) diabetic animals. The JCR:LA- cp rat is a model for type II non-insulin-dependent diabetes mellitus (NIDDM). This rat is insulin resistant, obese, and has high levels of circulating glucose, cholesterol, insulin, and triglycerides. The purpose of this study was to determine whether changes in cardiac myofibrillar, SR, and cardiomyocyte function exist in this model of type II diabetes. Myofibrils and SR were isolated from hearts by differential centrifugation. Surprisingly, we found that myofibrillar Ca2+-ATPase activities were unaltered in these animals. Ca2+uptake in isolated SR fractions was increased in diabetic cp/ cprats, whereas Ca2+-ATPase activity and ryanodine binding were unchanged. Cardiomyocytes isolated from hearts of control and experimental animals had similar active cell shortening and intracellular Ca2+concentration under basal conditions and in response to caffeine. Our data argue against the presence of a cardiomyopathy in this diabetic model and suggest that insulin may be an important factor in the cardiomyopathy observed in type I diabetic models.

The oxidative modification of low-density lipoprotein (LDL) plays an important role in atherosclerosis. Protecting LDL from oxidation has been shown to reduce the risk of coronary heart disease. In this study, we compared the protective effects of two lipophilic antioxidants (vitamin E and lazaroid) with two hydrophilic antioxidants (trolox and vitamin C) in the presence of several different free radical generating systems. Vitamin E (IC50 = 5.9 microM) and lazaroid (IC50 = 5.0 microM) were more effective in inhibiting lipid peroxidation caused by a Fe-ADP free radical generating system than vitamin C (IC50 = 5.2 x 10(3) microM) and trolox (IC5 = 1.2 x 10(3) microM). Preincubation of lipoproteins with a lipophilic antioxidant increased the protective effect against various free radicals. Preincubation with hydrophilic antioxidants did not have an effect. We also tested the efficacy of the antioxidants when the free radicals were generated within the lipid or the aqueous environment surrounding the LDL. For this purpose, we used the peroxyl generating azo-compounds AMVN (2,2'-azobis(2,4-dimethylvaleronitrile)) and AAPH (2,2'azobis(2-amidinopropane) dihydrochloride). All of the antioxidants tested were more effective against free radicals generated in a water soluble medium than they were against free radicals generated in a lipid environment. In conclusion, our data demonstrate that lipid solubility is an important factor for both the antioxidant and the free radical generating systems in determining the extent of lipid peroxidation in LDL. Our data also demonstrate that antioxidant efficacy in one set of experimental conditions may not necessarily translate into a similar degree of protection in another set of conditions where lipophilicity is a variable.

Ischemic heart disease is a significant problem in the diabetic population. Animal models of diabetes show a paradoxical resistance to ischemic challenge. The present treatise will discuss the mechanics involved and the central role that Na+-H+ exchanger plays in this response to ischemic-reperfusion injury.