Bacteria and the growing threat of multidrug resistance for invasive cardiac interventions.

Invasive cardiovascular procedures which include heart transplantations, congenital heart surgery, coronary artery bypass grafts, cardiac valve repair and replacement, and interventional cardiac electrophysiology procedures represent common mechanisms to treat a variety of cardiovascular diseases across the globe. The majority of these invasive approaches employ antibiotics as a regular and obligatory feature of the invasive procedure. Although the growing incidence of bacterial resistance to currently used antibiotics threatens to curtail the use of all interventional surgical techniques, it remains an underappreciated threat within the arsenal of cardiovascular therapies. It is reasonable to expect that the continued overuse of antibiotics and the frequent management of coronavirus disease 2019 (COVID-19) infected patients with high doses of antibiotics will inevitably accentuate the rise of multidrug resistance. The purpose of this article is to heighten awareness of the role of bacterial infections in cardiovascular disease, the use of antibiotics in today's cardiovascular surgical theaters, the threat facing cardiovascular surgery should multidrug resistance continue to rise unabated, and the development of new antibiotic platforms to solve this problem.

Oxidized phosphatidylcholines trigger ferroptosis in cardiomyocytes during ischemia-reperfusion injury.

Myocardial ischemia-reperfusion (I/R) injury increases the generation of oxidized phosphatidylcholines (OxPCs), which results in cell death. However, the mechanism by which OxPCs mediate cell death and cardiac dysfunction is largely unknown. The aim of this study was to determine the mechanisms by which OxPC triggers cardiomyocyte cell death during reperfusion injury. Adult rat ventricular cardiomyocytes were treated with increasing concentrations of various purified fragmented OxPCs. Cardiomyocyte viability, bioenergetic response, and calcium transients were determined in the presence of OxPCs. Five different fragmented OxPCs resulted in a decrease in cell viability, with 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) having the most potent cardiotoxic effect in both a concentration and time dependent manner (P < 0.05). POVPC and PONPC also caused a significant decrease in Ca2+ transients and net contraction in isolated cardiomyocytes compared to vehicle treated control cells (P < 0.05). PONPC depressed maximal respiration rate (P < 0.01; 54%) and spare respiratory capacity (P < 0.01; 54.5%). Notably, neither caspase 3 activation or TUNEL staining was observed in cells treated with either POVPC or PONPC. Further, cardiac myocytes treated with OxPCs were indistinguishable from vehicle-treated control cells with respect to nuclear high-mobility group box protein 1 (HMGBP1) activity. However, glutathione peroxidase 4 activity was markedly suppressed in cardiomyocytes treated with POVPC and PONPC coincident with increased ferroptosis. Importantly, cell death induced by OxPCs could be suppressed by E06 Ab, directed against OxPCs or by ferrostatin-1, which bound the sn-2 aldehyde of POVPC during I/R. The findings of the present study demonstrate that oxidation of phosphatidylcholines during I/R generate bioactive phospholipid intermediates that disrupt mitochondrial bioenergetics and calcium transients and provoke wide spread cell death through ferroptosis. Neutralization of OxPC with E06 or with ferrostatin-1 prevents cell death during reperfusion. Our study demonstrates a novel signaling pathway that operationally links generation of OxPC during cardiac I/R to ferroptosis. Interventions designed to target OxPCs may prove beneficial in mitigating ferroptosis during I/R injury in individuals with ischemic heart disease.NEW & NOTEWORTHY Oxidized phosphatidylcholines (OxPC) generated during reperfusion injury are potent inducers of cardiomyocyte death. Our studies have shown that OxPCs exert this effect through a ferroptotic process that can be attenuated. A better understanding of the OxPC cell death pathway can prove a novel strategy for prevention of cell death during myocardial reperfusion injury.

Cardioprotective Effects of Dietary Flaxseed Post-Infarction Are Associated with Changes in MicroRNA Expression.

MicroRNAs (miRNAs/miRs) such as miR-1, miR-133a, miR-133b, miR-135a, and miR-29b play a key role in many cardiac pathological remodeling processes, including apoptosis, fibrosis, and arrhythmias, after a myocardial infarction (MI). Dietary flaxseed has demonstrated a protective effect against an MI. The present study was carried out to test the hypothesis that dietary flaxseed supplementation before and after an MI regulates the expression of above-mentioned miRNAs to produce its cardioprotective effect. Animals were randomized after inducing MI by coronary artery ligation into: (a) sham MI with normal chow, (b) MI with normal chow, and (c-e) MI supplemented with either 10% milled flaxseed, or 4.4% flax oil enriched in alpha-linolenic acid (ALA), or 0.44% flax lignan secoisolariciresinol diglucoside. The feeding protocol consisted of 2 weeks before and 8 weeks after the surgery. Dietary flax oil supplementation selectively upregulated the cardiac expression of miR-133a, miR-135a, and miR-29b. The levels of collagen I expression were reduced in the flax oil group. We conclude that miR-133a, miR-135a, and miR-29b are sensitive to dietary flax oil, likely due to its rich ALA content. The cardioprotective effect of flaxseed in an MI could be due to modulation of these miRNAs.

Heat shock protein 60 involvement in vascular smooth muscle cell proliferation.

AIM: Heat shock protein 60 (Hsp60) is a mediator of stress-induced vascular smooth muscle cell (VSMC) proliferation. This study will determine, first, if the mitochondrial or cytoplasmic localization of Hsp60 is critical to VSMC proliferation and, second, the mechanism of Hsp60 induction of VSMC proliferation with a focus on modification of nucleocytoplasmic trafficking. METHODS AND RESULTS: Hsp60 was overexpressed in primary rabbit VSMCs with or without a mitochondrial targeting sequence (AdHsp60mito-). Both interventions induced an increase in VSMC PCNA expression and proliferation. The increase in VSMC PCNA expression and growth was not observed after siRNA-mediated knockdown of Hsp60 expression. Nuclear protein import in VSMC was measured by fluorescent microscopy using a microinjected fluorescent import substrate. Nuclear protein import was stimulated by both AdHsp60 and AdHsp60mito- treatments. AdHsp60 treatment also induced increases in nucleoporin (Nup) 62, Nup153, importin-α, importin-ß and Ran expression as well as cellular ATP levels compared to control. AdHsp60mito- treatment induced an up-regulation in importin-α, importin-ß and Ran expression compared to control. Hsp60 knockdown did not change nuclear protein import nor the expression of any nuclear transport receptors or nucleoporins. Both heat shock treatment and Hsp60 overexpression promoted the interaction of Ran with Hsp60. CONCLUSIONS: VSMC proliferation can be modulated via an Hsp60 dependent, cytosol localized mechanism that in part involves a stimulation of nuclear protein import through an interaction with Ran. This novel cellular signaling role for Hsp60 may be important in growth-based vascular pathologies like atherosclerosis and hypertension.

Na+-NQR (Na+-translocating NADH:ubiquinone oxidoreductase) as a novel target for antibiotics.

The recent breakthrough in structural studies on Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae creates a perspective for the systematic design of inhibitors for this unique enzyme, which is the major Na+ pump in aerobic pathogens. Widespread distribution of Na+-NQR among pathogenic species, its key role in energy metabolism, its relation to virulence in different species as well as its absence in eukaryotic cells makes this enzyme especially attractive as a target for prospective antibiotics. In this review, the major biochemical, physiological and, especially, the pharmacological aspects of Na+-NQR are discussed to assess its 'target potential' for drug development. A comparison to other primary bacterial Na+ pumps supports the contention that NQR is a first rate prospective target for a new generation of antimicrobials. A new, narrowly targeted furanone inhibitor of NQR designed in our group is presented as a molecular platform for the development of anti-NQR remedies.

Development of a novel rationally designed antibiotic to inhibit a nontraditional bacterial target.

The search for new nontraditional targets is a high priority in antibiotic design today. Bacterial membrane energetics based on sodium ion circulation offers potential alternative targets. The present work identifies the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR), a key respiratory enzyme in many microbial pathogens, as indispensible for the Chlamydia trachomatis infectious process. Infection by Chlamydia trachomatis significantly increased first H+ and then Na+ levels within the host mammalian cell. A newly designed furanone Na+-NQR inhibitor, PEG-2S, blocked the changes in both H+ and Na+ levels induced by Chlamydia trachomatis infection. It also inhibited intracellular proliferation of Chlamydia trachomatis with a half-minimal inhibitory concentration in the submicromolar range but did not affect the viability of mammalian cells or bacterial species representing benign intestinal microflora. At low nanomolar concentrations (IC50 value = 1.76 nmol/L), PEG-2S inhibited the Na+-NQR activity in sub-bacterial membrane vesicles isolated from Vibrio cholerae. Taken together, these results show, for the first time, that Na+-NQR is critical for the bacterial infectious process and is susceptible to a precisely targeted bactericidal compound in situ. The obtained data have immediate relevance for many different diseases caused by pathogenic bacteria that rely on Na+-NQR activity for growth, including sexually transmitted, pulmonary, oral, gum, and ocular infections.

The effect of flaxseed dose on circulating concentrations of alpha-linolenic acid and secoisolariciresinol diglucoside derived enterolignans in young, healthy adults.

PURPOSE: The primary endpoint was to determine the plasma concentration of alpha-linolenic acid (ALA), and its metabolites, following milled flaxseed consumption at four doses. Secondary outcomes focused on plasma enterolignan concentrations and the effects on tolerability, platelet aggregation, plasma lipids and urinary thromboxane levels. METHODS: Healthy, younger adults (n = 34; 18-49 years old) were randomized into four groups consuming one muffin daily for 30 days fortified with 10, 20, 30 or 40 g of milled flaxseed. Blood and urine were collected at baseline and 4 weeks. RESULTS: Plasma ALA concentrations increased with all flaxseed doses (P < 0.01), except the 20 g/day dose (P = 0.10), yet there was no significant dose-dependent response (P = 0.81). Only with the 30 g/day diet were n-3 polyunsaturated fatty acids (P = 0.007), and eicosapentaenoic acid (EPA) (P = 0.047) increased from baseline values. Docosapentaenoic acid and docosahexaenoic acid were not detected at any dose. Plasma total enterolignan concentrations significantly increased over time in all treatment groups, yet despite a dose-dependent tendency, no between-group differences were detected (P = 0.22). Flaxseed was well tolerated, even at the highest dose, as there were no reported adverse events, changes in cholesterol, platelet aggregation or urinary 11-dehydro-thromboxane B2. CONCLUSIONS: In healthy, younger adults, 10 g/day of milled flaxseed consumption is sufficient to significantly increase circulating ALA and total enterolignan concentrations; however, 30 g/day is required to convert ALA to EPA. Although all doses were well tolerated, 40 g/day is too low to attenuate cholesterol in this population.

Flaxseed consumption reduces blood pressure in patients with hypertension by altering circulating oxylipins via an α-linolenic acid-induced inhibition of soluble epoxide hydrolase.

UNLABELLED: In a randomized, double-blinded, controlled clinical trial, participants with peripheral arterial disease (75% hypertensive) consumed 30 g of milled flaxseed/d for 6 months. The flaxseed group exhibited significant reductions in systolic (-10 mm Hg) and diastolic (-7 mm Hg) blood pressure. Flaxseed contains the n3 fatty acid α-linolenic acid. Plasma α-linolenic acid increased with ingestion of flaxseed and was inversely associated with blood pressure. However, the antihypertensive mechanism was unclear. Oxylipins derived from polyunsaturated fatty acids regulate vascular tone. Therefore, the objective was to examine whether flaxseed consumption altered plasma oxylipins in a manner that influenced blood pressure. Plasma of FlaxPAD (Flaxseed for Peripheral Arterial Disease) participants underwent solid phase extraction and high-performance liquid chromatography-mass spectrometry/mass spectrometry analysis. The flaxseed group exhibited significant decreases in 8 plasma oxylipins versus control. Six of these (5,6-, 8,9-, 11,12-, 14,15-dihydroxyeicosatrienoic acid and 9,10- and 12,13-dihydroxyoctadecenoic acid) were products of soluble epoxide hydrolase, a pharmacological target for antihypertensive treatment. Patients exhibiting a decrease in total plasma soluble epoxide hydrolase-derived oxylipins, exhibited a significant decrease in systolic blood pressure (mean [95% confidence interval], -7.97 [-14.4 to -1.50] mm Hg) versus those who exhibited increased plasma soluble epoxide hydrolase-derived oxylipins (+3.17 [-4.78 to 11.13] mm Hg). These data suggest that a flaxseed bioactive may have decreased blood pressure via soluble epoxide hydrolase inhibition. Using a soluble epoxide hydrolase inhibitor screening assay, increasing concentrations of α-linolenic acid decreased soluble epoxide hydrolase activity (P=0.0048; ρ=-0.94). In conclusion, α-linolenic acid in flaxseed may have inhibited soluble epoxide hydrolase, which altered oxylipin concentrations that contributed to the antihypertensive effects in patients with peripheral arterial disease. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT00781950.

Potent antihypertensive action of dietary flaxseed in hypertensive patients.

Flaxseed contains ω-3 fatty acids, lignans, and fiber that together may provide benefits to patients with cardiovascular disease. Animal work identified that patients with peripheral artery disease may particularly benefit from dietary supplementation with flaxseed. Hypertension is commonly associated with peripheral artery disease. The purpose of the study was to examine the effects of daily ingestion of flaxseed on systolic (SBP) and diastolic blood pressure (DBP) in peripheral artery disease patients. In this prospective, double-blinded, placebo-controlled, randomized trial, patients (110 in total) ingested a variety of foods that contained 30 g of milled flaxseed or placebo each day over 6 months. Plasma levels of the ω-3 fatty acid α-linolenic acid and enterolignans increased 2- to 50-fold in the flaxseed-fed group but did not increase significantly in the placebo group. Patient body weights were not significantly different between the 2 groups at any time. SBP was ≈ 10 mm Hg lower, and DBP was ≈ 7 mm Hg lower in the flaxseed group compared with placebo after 6 months. Patients who entered the trial with a SBP ≥ 140 mm Hg at baseline obtained a significant reduction of 15 mm Hg in SBP and 7 mm Hg in DBP from flaxseed ingestion. The antihypertensive effect was achieved selectively in hypertensive patients. Circulating α-linolenic acid levels correlated with SBP and DBP, and lignan levels correlated with changes in DBP. In summary, flaxseed induced one of the most potent antihypertensive effects achieved by a dietary intervention.

Effects of dietary flaxseed on atherosclerotic plaque regression.

Dietary flaxseed can retard the progression of atherosclerotic plaques. However, it remains unclear whether these antiatherogenic effects extend to plaque regression. In the present study, the therapeutic potential of dietary flaxseed on atherosclerotic plaque regression and vascular contractile function was evaluated using a novel rabbit model. Rabbits were randomly assigned to receive either a regular diet for 12 wk (group I) or a 1% cholesterol-supplemented diet for 4 wk followed by a regular diet for 8 wk (group II). The remaining experimental animals were treated as in group II but were fed for an additional 14 wk with either a regular diet (group III) or a 10% flaxseed-supplemented diet (group IV). Animals in group II showed clear evidence of plaque growth stabilization. Their vessels also exhibited significantly lower norepinephrine-induced contraction and an impaired relaxation response to acetylcholine compared with animals in group I. Dietary flaxseed supplementation resulted in a significant ≈40% reduction in plaque formation (P = 0.033). Animals in both groups II and III displayed improved contraction and endothelium-dependent vessel relaxation. Dietary flaxseed is a valuable strategy to accelerate the regression of atherosclerotic plaques; however, flaxseed intervention did not demonstrate a clear beneficial effect on the vessel contractile response and endothelium-dependent vasorelaxation.

Oxidized LDL enhances stretch-induced smooth muscle cell proliferation through alterations in nuclear protein import.

Mechanical stress contributes to hypertension and atherosclerosis partly through the stimulation of vascular smooth muscle cell (VSMC) proliferation. Oxidized low density lipoprotein (oxLDL) is another important atherogenic factor that can increase VSMC proliferation. The purpose of this study was to investigate whether oxLDL could further enhance the proliferative action of mechanical stretch on VSMC, and to determine the mechanism responsible for this interaction. Because nuclear protein import is critical in regulating gene expression, transcription, and cell proliferation, its involvement in the mitogenic effects of oxLDL and mechanical stress was studied. OxLDL enhanced the proliferative effects of mechanical stretch on its own in rabbit aortic VSMC, and induced increases in the expression of HSP60 in an additive manner. Adenoviral-mediated overexpression of HSP60 induced increases in cell proliferation compared with uninfected VSMC. Mechanical stretch and oxLDL stimulated the rate of nuclear protein import in VSMC and increased the expression of nucleoporins. These effects were sensitive to inhibition of the MAPK pathway. We conclude that oxLDL and mechanical stretch have a synergistic effect on VSMC proliferation. This synergistic effect is induced through a stimulation of nuclear protein import via HSP60 and an activation of the MAPK pathway.

Chlamydophila pneumoniae infection induces alterations in vascular contractile responses.

Chlamydophila pneumoniae infection has been associated in previous studies with coronary artery disease. The live bacterium has been detected within atherosclerotic plaques and can induce the structural remodeling of the vessel wall. However, the direct effects of infection on the contractile characteristics of the arteries remain unknown. Left anterior descending coronary arteries isolated from porcine hearts were dissected and placed in culture medium for 72 hours before infection with C. pneumoniae. Contractile responses to high molar KCl and u46619 levels and relaxation responses to bradykinin and sodium nitroprusside were assessed at days 5 and 10 postinfection. C. pneumoniae induced decreases in both KCl- and u46619-induced contractile responses at both time points. The altered contractile responses coincided with a down-regulation of L-type Ca(2+) channels at both time points and inositol 1,4,5-triphosphate receptor (IP3R) levels at day 10 postinfection. Infection also induced attenuation of the endothelial-dependent relaxation response to bradykinin at day 10 postinfection. A decrease in endothelial nitric oxide synthase expression levels was noted at day 10 postinfection. Furthermore, an increase in superoxide production combined with an increase in p22phox expression levels was also observed at this time point. These findings indicate that C. pneumoniae infection can directly alter the vascular contractile responses in porcine coronary arteries, providing additional evidence for the role of C. pneumoniae infection in cardiovascular disease.

Oxidized LDL promotes the mitogenic actions of Chlamydia pneumoniae in vascular smooth muscle cells.

AIMS: The atherogenic actions of Chlamydia pneumoniae (C. pneumoniae), a common respiratory pathogen, are dependent upon a high-cholesterol environment in vivo. It is possible that oxidized low-density lipoprotein (oxLDL) is responsible for promoting the atherogenic effects of C. pneumoniae through a stimulation of cell proliferation. This study determined whether oxLDL can enhance the mitogenic action of C. pneumoniae in vascular smooth muscle cells (VSMCs) and the involvement of mitogen-activated protein kinase (MAPK) pathways and heat shock protein 60 (HSP60) in these mechanisms. METHODS AND RESULTS: Primary rabbit VSMCs were treated with live C. pneumoniae, heat-inactivated C. pneumoniae or infection medium, and subsequently incubated for up to 48 h in the presence or absence of oxLDL. Chlamydia pneumoniae infection alone stimulated cell proliferation and the addition of oxLDL significantly amplified this proliferative effect. This proliferation was accompanied by extracellular signal-regulated kinase-1 and -2 (ERK1/2) activation and an up-regulation of HSP60 expression. Changes in proliferation and HSP60 expression were attenuated by the inhibition of ERK1/2. CONCLUSION: These results indicate a novel role for oxLDL in promoting the mitogenic actions of C. pneumoniae in the vasculature. ERK1/2 is an important factor in the stress-mediated response and HSP60 up-regulation in VSMC. These data provide mechanistic evidence that C. pneumoniae may stimulate atherogenesis.

The alpha linolenic acid content of flaxseed is associated with an induction of adipose leptin expression.

Dietary flaxseed has cardioprotective effects that may be achieved through its rich content of the omega-3 fatty acid, alpha linolenic acid (ALA). Because ALA can be stored in adipose tissue, it is possible that some of its beneficial actions may be due to effects it has on the adipose tissue. We investigated the effects of dietary flaxseed both with and without an atherogenic cholesterol-enriched diet to determine the effects of dietary flaxseed on the expression of the adipose cytokines leptin and adiponectin. Rabbits were fed one of four diets: a regular (RG) diet, or a regular diet with added 0.5% cholesterol (CH), or 10% ground flaxseed (FX), or both (CF) for 8 weeks. Levels of leptin and adiponectin expression were assessed by RT-PCR in visceral adipose tissue. Consumption of flaxseed significantly increased plasma and adipose levels of ALA. Leptin protein and mRNA expression were lower in CH animals and were elevated in CF animals. Changes in leptin expression were strongly and positively correlated with adipose ALA levels and inversely correlated with levels of en face atherosclerosis. Adiponectin expression was not significantly affected by any of the dietary interventions. Our data demonstrate that the type of fat in the diet as well as its caloric content can specifically influence leptin expression. The findings support the hypothesis that the beneficial cardiovascular effects associated with flaxseed consumption may be related to a change in leptin expression.

Reduced expression of the Na+/Ca2+ exchanger in adult cardiomyocytes via adenovirally delivered shRNA results in resistance to simulated ischemic injury.

The Na(+)/Ca(2+) exchanger (NCX) is proposed to be an important protein in the regulation of Ca(2+) movements in the heart. This Ca(2+) regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca(2+) overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to approximately 60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. Ca(2+) homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic Ca(2+) and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes.

Chlamydophila pneumoniae infection leads to smooth muscle cell proliferation and thickening in the coronary artery without contributions from a host immune response.

Chlamydophila pneumonia (C. pneumonia) infection has been associated with the progression of atherosclerosis. It remains unclear, however, whether C. pneumoniae in the absence of an immune response can alone initiate atherogenic events within a complex vessel environment. Left anterior descending coronary arteries isolated from porcine hearts were dissected and placed in culture medium for 72 hours before infection with C. pneumoniae. C. pneumoniae replicated within the arterial wall for the duration of the experiment (up to 10 days). A significant increase in chlamydial-HSP60 protein expression from day 2 to 10 post-infection (pi) indicated the presence of metabolically active C. pneumonia within infected vessels. Significant arterial thickening in infected coronary segments was observed by a considerable decrease in the ratio of lumen to total vessel area (48 +/- 3% at day 4 pi versus 23 +/- 3% at day 10 pi) and a significant increase in the ratio of media to luminal area (113 +/- 16% at day 4 pi versus 365 +/- 65% at day 10 pi). Structural changes were accompanied by an up-regulation of host HSP60 and proliferating cell nuclear antigen expression levels. Immunohistochemical staining confirmed proliferating cell nuclear antigen expression to be primarily localized within smooth muscle cells of the medial area. These results demonstrate that C. pneumoniae infection can stimulate arterial thickening in a complex vessel environment without the presence of a host immune response and further supports the involvement of HSP60 in this action.

Trans-fatty acids in the diet stimulate atherosclerosis.

Epidemiological evidence has associated dietary trans-fatty acids (TFAs) with coronary heart disease. It is assumed that TFAs stimulate atherosclerosis, but this has not been proven. The purpose of this study was to determine the effects of consuming 2 concentrations of TFAs obtained from commercially hydrogenated vegetable shortening on atherosclerotic development in the presence or absence of elevated dietary cholesterol. Low-density lipoprotein receptor-deficient mice were fed 1 of 7 experimental diets for 14 weeks: low regular fat (LR), low trans-fat (LT), regular high fat, high trans-fat (HT), or a diet containing 2% cholesterol with low regular fat (C + LR), low trans-fat (C + LT), or high trans-fat (C + HT). The extent of lesion development was quantified by en face examination of the dissected aortae. Dietary cholesterol supplementation significantly elevated serum cholesterol levels. Surprisingly, this rise was partially attenuated by the addition of TFAs (C + LT and C + HT) in the diet. Serum triglyceride levels were elevated with the higher-fat diets and with the combination of trans-fat and cholesterol. Animals consuming TFAs in the absence of dietary cholesterol developed a significantly greater extent of aortic atherosclerotic lesions as compared with control animals (LT > LR and HT > regular high fat). Atherosclerotic lesions were more extensive after cholesterol feeding, but the addition of TFAs to this atherogenic diet did not advance atherosclerotic development further. In summary, TFAs are atherogenic on their own; but they do not stimulate further effects beyond the strongly atherogenic effects of dietary cholesterol.

Oxidized LDL affects smooth muscle cell growth through MAPK-mediated actions on nuclear protein import.

Oxidized low density lipoprotein (oxLDL) plays an important role in the development of atherosclerosis partly through an action on cell proliferation and cell apoptosis. Nuclear protein import (NPI) is critical in regulating gene expression, transcription, and subsequently cell proliferation and apoptosis. The aim of this study was to determine if exposure of vascular smooth muscle cells (VSMC) to oxLDL affects cell growth by inducing alterations in NPI and nuclear pore density. VSMC were exposed for different times to oxLDL. Cells were then injected with a protein import substrate (Alexa488-BSA-NLS) to visually monitor nuclear transport with the confocal microscope. The effect of MAPK inhibitors (SB203580 and PD98059) was investigated and western immunoblottings were also performed. Shorter exposure times of VSMC to oxLDL, but not to native LDL, significantly increased NPI, nuclear pore expression (p62), PCNA expression, and cell number. These changes occurred through an ERK MAPK-dependent mechanism. However, longer exposures to oxLDL decreased NPI, nuclear pore expression, and increased apoptosis marker (cleaved PARP) expression through a p38 MAPK-dependent mechanism. We conclude that limited exposure to oxLDL may influence cell proliferation and apoptosis through an action on nucleocytoplasmic trafficking. The nucleus and NPI may represent a novel therapeutic target to control diseases like atherosclerosis that have changes in cell growth as a central feature.

A comparison of fish oil, flaxseed oil and hempseed oil supplementation on selected parameters of cardiovascular health in healthy volunteers.

OBJECTIVE: The impact of dietary polyunsaturated fatty acids (PUFAs) of the n-6 and n-3 series on the cardiovascular system is well documented. To directly compare the effects of three dietary oils (fish, flaxseed and hempseed) given in concentrations expected to be self-administered in the general population on specific cardiovascular parameters in healthy volunteers. DESIGN: 86 healthy male and female volunteers completed a 12 week double blinded, placebo controlled, clinical trial. They were randomly assigned to one of the four groups. Subjects were orally supplemented with two 1 gm capsules of placebo, fish oil, flaxseed oil or hempseed oil per day for 12 weeks. RESULTS: Plasma levels of the n-3 fatty acids docosahexanoic acid and eicosapentanoic acid increased after 3 months supplementation with fish oil. Alpha linolenic acid concentrations increased transiently after flaxseed supplementation. However, supplementation with hempseed oil did not significantly alter the concentration of any plasma fatty acid. The lipid parameters (TC, HDL-C, LDL-C and TG) did not show any significant differences among the four groups. Oxidative modification of LDL showed no increase in lag time over the 12 wk period. None of the dietary interventions induced any significant change in collagen or thrombin stimulated platelet aggregation and no increase in the level of inflammatory markers was observed. CONCLUSION: From a consumer's perspective, ingesting 2 capsules of any of these oils in an attempt to achieve cardiovascular health benefits may not provide the desired or expected result over a 3 month period.

Ceramide regulation of nuclear protein import.

Nucleocytoplasmic trafficking is an essential and responsive cellular mechanism that directly affects cell growth and proliferation, and its potential to address metabolic challenge is incompletely defined. Ceramide is an antiproliferative sphingolipid found within vascular smooth muscle cells in atherosclerotic plaques, but its mechanism of action remains unclear. The hypothesis that ceramide inhibits cell growth through nuclear transport regulation was tested. In smooth muscle cells, exogenously supplemented ceramide inhibited classical nuclear protein import that involved the activation of cytosolic p38 mitogen-activated protein kinase (MAPK). After application of SB 202190, a specific and potent pharmacological antagonist of p38 MAPK, sphingolipid impingement on nuclear transport was corrected. Distribution pattern assessments of two essential nuclear transport proteins, importin-alpha and Cellular Apoptosis Susceptibility, revealed ceramide-mediated relocalization that was reversed upon the addition of SB 202190. Furthermore, cell counts, nuclear cyclin A, and proliferating cell nuclear antigen expression, markers of cellular proliferation, were diminished after ceramide treatment and effectively rescued by the addition of inhibitor. Together, these data demonstrate, for the first time, the sphingolipid regulation of nuclear import that defines and expands the adaptive capacity of the nucleocytoplasmic transport machinery.