Phytosterol-enriched foods are increasingly marketed to lower cholesterol levels and atherosclerosis in the general population. Phytosterols reduce cholesterol absorption, but the molecular mechanism ...is controversial. We therefore investigated the phytosterol effects on cholesterol metabolism in human enterocyte, hepatocyte, and macrophage models relevant for sterol absorption, reverse transport, and excretion. Isomolar sitosterol (50 μmol/L) was less effectively taken up by enterocytes than cholesterol but suppressed apical cholesterol uptake by 50% (P < 0.01) and basolateral secretion by two-thirds (P < 0.01) whether added in micelles or ethanol or complexed to cyclodextrin. In contrast, enterocytes handled nanomolar (3)H-sitosterol similarly to cholesterol. Enterocytes selectively oxidized all sterols to 27-hydroxy- and 27-carboxy-sterols. Conversion rates were much lower for sitosterol (0.05 ± 0.02 nmol/mg protein) and campesterol (0.48 ± 0.10) compared with cholesterol (3.73 ± 0.60) (P < 0.001). 27-Hydroxycholesterol (27OH-C) activated liver-X-receptor alpha (LXRα) (P < 0.01) and stimulated ATP-binding cassette transporter (ABC) A1 expression (P < 0.001) and basolateral systemic cholesterol secretion from enterocytes (P < 0.05). In co-incubations, phytosterols inhibited 27OH-C generation by sterol 27-hydroxylase (P < 0.001) and reduced LXRα-mediated ABCA1 expression (P < 0.01) and basolateral systemic cholesterol secretion. In contrast, ABCG8 transcription and apical sterol resecretion was unchanged by LXRα activation in human enterocytes. Exogenous LXRα agonists reverted sterol selectivity and phytosterol cholesterol interaction. Due to constitutive apical expression of ABCG5/G8 and LXRα-enhanced basolateral expression of ABCA1 in enterocytes, interference of phytosterols with the generation of the dominating LXRα-agonist 27OH-C blocks the self-priming component of cholesterol absorption. This local LXRα antagonism of dietary phytosterols contributes to sterol selectivity and reduces fractional cholesterol absorption and preloading of nascent HDL with dietary cholesterol.
Niacin, the first lipid lowering drug shown to improve survival after myocardial infarction, decreases LDL and increases HDL cholesterol levels. These effects cannot fully be explained by its ...suspected mechanism of action, inhibition of lipolysis and hepatic VLDL synthesis. Niacin has also been shown to interfere with the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and massively stimulate prostaglandin D
2 (PGD
2) formation. The major metabolite of PGD
2, 15-deoxy-Δ
12,14-prostaglandin J
2 (15d-PGJ
2), was recently identified as the most potent endogenous PPARγ activator. We, therefore, studied the effects of niacin on the PPARγ- and cAMP-dependent expression of receptors promoting reverse cholesterol transport. The transcription of PPARγ-, HDL-, LDL- and scavenger-receptors and the sterol exporter ABCA1, were measured by quantitative RT-PCR and cellular cholesterol efflux and PPARγ activation studied in macrophage and hepatocyte models. Niacin stimulated the translocation of PPARγ and the transcription of PPARγ, CD36 and ABCA1 in monocytoid cells, whereas the LDL-receptor (LDL-R) was unchanged. Thereby niacin enhanced HDL-mediated cholesterol efflux from the cells resulting in a reduced cellular cholesterol content. The niacin effect on CD36 but not on ABCA1 was prevented by cyclooxygenase inhibition, whereas the niacin effect on ABCA1 but not on CD36 was prevented by PKA inhibition, suggesting mediation by the 15d-PGJ
2/PPARγ and the cAMP/PKA pathways, respectively. These new actions of niacin on several key effectors of reverse cholesterol transport out of the vessel wall provide a rational to expect regression of atherosclerosis and test the combination of niacin with statins for an overadditive clinical benefit.
Methylphenidate (MPH) is the first-line pharmacological treatment of attention-deficit/hyperactivity disorder (ADHD). MPH binds to the dopamine (DA) transporter (DAT), which has high density in the ...striatum. Assessments of the striatal dopamine transporter by single positron emission computed tomography (SPECT) in childhood and adolescent patients are rare but can provide insight on how the effects of MPH affect DAT availability. The aim of our within-subject study was to investigate the effect of MPH on DAT availability and how responsivity to MPH in DAT availability is linked to clinical symptoms and cognitive functioning.
Thirteen adolescent male patients (9-16 years) with a diagnosis of ADHD according to the DSM-IV and long-term stimulant medication (for at least 6 months) with MPH were assessed twice within 7 days using SPECT after application of I-123-β-CIT to examine DAT binding potential (DAT BP). SPECT measures took place in an on- and off-MPH status balanced for order across participants. A virtual reality continuous performance test was performed at each time point. Further clinical symptoms were assessed for baseline off-MPH.
On-MPH status was associated with a highly significant change (-29.9%) of striatal DAT BP as compared to off-MPH (
= -4.12,
= 0.002). A more pronounced change in striatal DAT BP was associated with higher off-MPH attentional and externalizing symptom ratings (Pearson
= 0.68,
= 0.01). Striatal DAT BP off-MPH, but not on-MPH, was associated with higher symptom ratings (Pearson
= 0.56,
= 0.04).
Our findings corroborate previous reports from mainly adult samples that MPH changes striatal DAT BP availability and suggest higher off-MPH DAT BP, likely reflecting low baseline DA levels, as a marker of symptom severity.
Marked anti-atheromatous effects of the anti-inflammatory cytokine interleukin-10 (IL-10) were observed in several lipid-driven animal models of arteriosclerosis. We therefore investigated whether ...IL-10 affects macrophage cholesterol handling.
Human THP-1 cells and peripheral monocytes served as macrophage models. Specific mRNA was quantified by real-time RT-PCR, protein expression by flow cytometry and Western blotting. Cellular cholesterol handling was studied by lipoprotein-facilitated uptake and efflux assays. IL-10 effects were also studied in cells transfected with liver X receptor alpha (LXRalpha)-siRNA or a LXRalpha response element (LXRE) reporter construct.
Picomolar IL-10 suppressed basal and peroxisome proliferator-activated receptor gamma (PPARgamma)-stimulated transcription of the scavenger receptor CD36 due to reduced PPARgamma protein expression. In contrast, IL-10 stimulated transcription of the active cellular cholesterol exporters ATP-binding cassette transporters A1 and G1 (ABCA1, ABCG1) and the LDL receptor, whereas scavenger receptor-BI (SR-BI) was unchanged. The reduction of CD36 and stimulation of ABCA1 expression was confirmed in human monocytes. Thereby, IL-10 prevented cellular cholesterol overloading from oxidized LDL (oxLDL) and enhanced efflux to apoA-containing particles initiating reverse cholesterol transport. Experiments with inhibitors, LXRalpha silencing and the LXRE reporter gene construct supported the proximal transmission of the IL-10 effect on ABCA1 by the IL-10 receptor/signal transducer and activator of transcription 3 (STAT3) pathway and distal cross-talk to the LXRalpha and PPARalpha/retinoic acid X receptor (RXR) and cAMP/protein kinase A (PKA) pathways.
In addition to immune and anti-inflammatory actions, IL-10 redirects macrophage cholesterol handling towards reverse cholesterol transport, which contributes to its anti-atherosclerotic action.
Type 2 diabetes (T2D) has a complex pathophysiology which makes modeling the disease difficult. We aimed to develop a novel model for simulating T2D in vitro, including hyperglycemia, hyperlipidemia, ...and variably elevated insulin levels targeting muscle cells. We investigated insulin resistance (IR), cellular respiration, mitochondrial morphometry, and the associated function in different T2D-mimicking conditions in rodent skeletal (C2C12) and cardiac (H9C2) myotubes. The physiological controls included 5 mM of glucose with 20 mM of mannitol as osmotic controls. To mimic hyperglycemia, cells were exposed to 25 mM of glucose. Further treatments included insulin, palmitate, or both. After short-term (24 h) or long-term (96 h) exposure, we performed radioactive glucose uptake and mitochondrial function assays. The mitochondrial size and relative frequencies were assessed with morphometric analyses using electron micrographs. C2C12 and H9C2 cells that were treated short- or long-term with insulin and/or palmitate and HG showed IR. C2C12 myotubes exposed to T2D-mimicking conditions showed significantly decreased ATP-linked respiration and spare respiratory capacity and less cytoplasmic area occupied by mitochondria, implying mitochondrial dysfunction. In contrast, the H9C2 myotubes showed elevated ATP-linked and maximal respiration and increased cytoplasmic area occupied by mitochondria, indicating a better adaptation to stress and compensatory lipid oxidation in a T2D environment. Both cell lines displayed elevated fractions of swollen/vacuolated mitochondria after T2D-mimicking treatments. Our stable and reproducible in vitro model of T2D rapidly induced IR, changes in the ATP-linked respiration, shifts in energetic phenotypes, and mitochondrial morphology, which are comparable to the muscles of patients suffering from T2D. Thus, our model should allow for the study of disease mechanisms and potential new targets and allow for the screening of candidate therapeutic compounds.
Background
GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI‐Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not ...increase bleeding in clinical studies. GPVI‐Fc is a potent inhibitor of atherosclerotic plaque‐induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI‐Fc to the ectonucleotidase CD39 (fusion protein GPVI‐CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion‐directed dual antiplatelet therapy that is expected to lack systemic bleeding risks.
Methods and Results
GPVI‐CD39 effectively stimulated local ADP degradation and, compared with GPVI‐Fc alone, led to significantly increased inhibition of ADP‐, collagen‐, and human plaque–induced platelet aggregation in Multiplate aggregometry and plaque‐induced platelet thrombus formation under arterial flow conditions. GPVI‐CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride–induced arterial thrombosis, GPVI‐CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo.
Conclusions
GPVI‐CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI‐Fc–mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk–benefit ratio.
Background
Public stroke awareness and knowledge may be supportive for stroke prevention and emergency care-seeking behavior after the acute event, which is highly important for early treatment ...onset.
Aims
In an urban population in Northern Germany (Hannover), a six-month stroke educational campaign was conducted. We expected an increase in stroke knowledge and awareness thereafter.
Methods
Computer-assisted telephone interviews were randomly conducted among 1004 representative participants before and 1010 immediately after the educational multimedia campaign. The computer-assisted telephone interviews focused on questions about stroke knowledge and interventions remembered.
Results
Knowledge of stroke risk factors increased during the campaign for overweight, physical inactivity, old age, and stroke in family (P < 0·05). The knowledge of stroke warning signs was low, although it significantly increased during the campaign (P < 0·001) as paresis/weakness (46%) and speech problems (31%) were most frequently named. The majority of respondents indicated that the first action after suffering from stroke should be calling emergency care (74% before vs. 84% after campaign, P < 0·001).
Conclusions
Our data indicate that stroke knowledge and awareness, which could provide earlier presentation to the emergency unit for timely treatment onset, are still low in urban Northern Germany but may decisively be increased by educational campaigns.
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