The impact of a summative national prescribing assessment and curriculum type on the development of the prescribing competence of junior doctors.

PURPOSE: The primary aim of this study was to investigate the effect of including the Dutch National Pharmacotherapy Assessment (DNPA) in the medical curriculum on the level and development of prescribing knowledge and skills of junior doctors. The secondary aim was to evaluate the relationship between the curriculum type and the prescribing competence of junior doctors. METHODS: We re-analysed the data of a longitudinal study conducted in 2016 involving recently graduated junior doctors from 11 medical schools across the Netherlands and Belgium. Participants completed three assessments during the first year after graduation (around graduation (+ / - 4 weeks), and 6 months, and 1 year after graduation), each of which contained 35 multiple choice questions (MCQs) assessing knowledge and three clinical case scenarios assessing skills. Only one medical school used the DNPA in its medical curriculum; the other medical schools used conventional means to assess prescribing knowledge and skills. Five medical schools were classified as providing solely theoretical clinical pharmacology and therapeutics (CPT) education; the others provided both theoretical and practical CPT education (mixed curriculum). RESULTS: Of the 1584 invited junior doctors, 556 (35.1%) participated, 326 (58.6%) completed the MCQs and 325 (58.5%) the clinical case scenarios in all three assessments. Junior doctors whose medical curriculum included the DNPA had higher knowledge scores than other junior doctors (76.7% [SD 12.5] vs. 67.8% [SD 12.6], 81.8% [SD 11.1] vs. 76.1% [SD 11.1], 77.0% [12.1] vs. 70.6% [SD 14.0], p < 0.05 for all three assessments, respectively). There was no difference in skills scores at the moment of graduation (p = 0.110), but after 6 and 12 months junior doctors whose medical curriculum included the DNPA had higher skills scores (both p < 0.001). Junior doctors educated with a mixed curriculum had significantly higher scores for both knowledge and skills than did junior doctors educated with a theoretical curriculum (p < 0.05 in all assessments). CONCLUSION: Our findings suggest that the inclusion of the knowledge focused DNPA in the medical curriculum improves the prescribing knowledge, but not the skills, of junior doctors at the moment of graduation. However, after 6 and 12 months, both the knowledge and skills were higher in the junior doctors whose medical curriculum included the DNPA. A curriculum that provides both theoretical and practical education seems to improve both prescribing knowledge and skills relative to a solely theoretical curriculum.

The Dutch list of essential drugs for undergraduate medical education: A modified Delphi study.

AIMS: Prescribing errors among junior doctors are common in clinical practice because many lack prescribing competence after graduation. This is in part due to inadequate education in clinical pharmacology and therapeutics (CP&T) in the undergraduate medical curriculum. To support CP&T education, it is important to determine which drugs medical undergraduates should be able to prescribe safely and effectively without direct supervision by the time they graduate. Currently, there is no such list with broad-based consensus. Therefore, the aim was to reach consensus on a list of essential drugs for undergraduate medical education in the Netherlands. METHODS: A two-round modified Delphi study was conducted among pharmacists, medical specialists, junior doctors and pharmacotherapy teachers from all eight Dutch academic hospitals. Participants were asked to indicate whether it was essential that medical graduates could prescribe specific drugs included on a preliminary list. Drugs for which ≥80% of all respondents agreed or strongly agreed were included in the final list. RESULTS: In all, 42 (65%) participants completed the two Delphi rounds. A total of 132 drugs (39%) from the preliminary list and two (3%) newly proposed drugs were included. CONCLUSIONS: This is the first Delphi consensus study to identify the drugs that Dutch junior doctors should be able to prescribe safely and effectively without direct supervision. This list can be used to harmonize and support the teaching and assessment of CP&T. Moreover, this study shows that a Delphi method is suitable to reach consensus on such a list, and could be used for a European list.

Do we become better prescribers after graduation: A 1-year international follow-up study among junior doctors.

AIM: The aim of this study was to investigate how the prescribing knowledge and skills of junior doctors in the Netherlands and Belgium develop in the year after graduation. We also analysed differences in knowledge and skills between surgical and nonsurgical junior doctors. METHODS: This international, multicentre (n = 11), longitudinal study analysed the learning curves of junior doctors working in various specialties via three validated assessments at about the time of graduation, and 6 months and 1 year after graduation. Each assessment contained 35 multiple choice questions (MCQs) on medication safety (passing grade ≥85%) and three clinical scenarios. RESULTS: In total, 556 junior doctors participated, 326 (58.6%) of whom completed the MCQs and 325 (58.5%) the clinical case scenarios of all three assessments. Mean prescribing knowledge was stable in the year after graduation, with 69% (SD 13) correctly answering questions at assessment 1 and 71% (SD 14) at assessment 3, whereas prescribing skills decreased: 63% of treatment plans were considered adequate at assessment 1 but only 40% at assessment 3 (P < .001). While nonsurgical doctors had similar learning curves for knowledge and skills as surgical doctors (P = .53 and P = .56 respectively), their overall level was higher at all three assessments (all P < .05). CONCLUSION: These results show that junior doctors' prescribing knowledge and skills did not improve while they were working in clinical practice. Moreover, their level was under the predefined passing grade. As this might adversely affect patient safety, educational interventions should be introduced to improve the prescribing competence of junior doctors.

Haemodynamic effects of the flavonoid quercetin in rats revisited.

BACKGROUND AND PURPOSE: The flavonoid quercetin increased the in vitro potency of the α1 -antagonist tamsulosin to reduce phenylephrine-dependent arterial contractions by 10-fold. To examine if this supplement-drug interaction luxates hypotensive and orthostatic events in vivo, several set of studies were conducted in spontaneously hypertensive (SHR) and normotensive (Wistar Kyoto [WKY]) rats. EXPERIMENTAL APPROACH: First, in rats pretreated with quercetin or its vehicle, responses to phenylephrine and tamsulosin were examined. Second, tamsulosin-induced changes in renal, mesenteric, hindquarter and carotid conductance were compared in quercetin- and vehicle-treated rats instrumented with Doppler flow probes. Animals were also placed on a tilt table to record regional haemodynamic changes to orthostatic challenges. Third, adult SHR were instrumented with telemeters to measure 24-hr patterns of BP. Recordings were made before and during a 5-week oral treatment of quercetin. Finally, pre-hypertensive SHR were treated with quercetin from 4 to 8 weeks of age and arterial pressure was measured at 8 and 12 weeks. KEY RESULTS: Pretreatment with quercetin did not influence the responses to phenylephrine and tamsulosin, in neither WKY nor SHR. While tamsulosin treatment and tilting lowered BP and increased conductance in all vascular beds, effect size was not influenced by pretreatment with quercetin. Prolonged treatment with quercetin, in either prehypertensive SHR or adult SHR with established hypertension did not lower BP. CONCLUSIONS AND IMPLICATIONS: Cumulatively, these data demonstrate that quercetin does not amplify haemodynamic effects of tamsulosin or tilting in vivo in rats and has no effect on BP development in SHR.

The Beneficial Effects of UM206 on Wound Healing After Myocardial Infarction in Mice Are Lost in Follow-Up Experiments.

Introduction: An inadequate wound healing following myocardial infarction (MI) is one of the main etiologies of heart failure (HF) development. Interventions aiming at improving this process may contribute to preserving cardiac function after MI. Our group, as well as others, have demonstrated the crucial role of Wnt/frizzled signaling in post-MI remodeling. In this overview, we provide the results of different studies aimed at confirming an initial study from our group, in which we observed beneficial effects of administration of a peptide fragment of Wnt5a, UM206, on infarct healing in a mouse MI model. Methods: Mice were subjected to permanent left coronary artery ligation, and treated with saline (control) or UM206, administered via osmotic minipumps. Cardiac function was assessed by echocardiography and hemodynamic measurements, while infarct size and myofibroblast content were characterized by (immuno)histochemistry. Results: In total, we performed seven follow-up studies, but we were unable to reproduce the beneficial effects of UM206 on infarct healing in most of them. Variations in dose and timing of UM206 administration, its manufacturer and the genetic background of the mice could not restore the phenotype. An in-depth analysis of the datasets revealed that the absence of effect of UM206 coincided with a lack of adverse cardiac remodeling and HF development in all experimental groups, irrespective of the treatment. Discussion: Irreproducibility of experimental observations is a major issue in biomedical sciences. It can arise from a relatively low number of experimental observations in the original study, a faulty hypothesis or a variation in the experimental model that cannot be controlled. In this case, the lack of adverse cardiac remodeling and lung weight increases in the follow-up studies point out to altered experimental conditions as the most likely explanation.

Angiotensin II-induced hypertension in rats is only transiently accompanied by lower renal oxygenation.

Activation of the renin-angiotensin system may initiate chronic kidney disease. We hypothesised that renal hypoxia is a consequence of hemodynamic changes induced by angiotensin II and occurs prior to development of severe renal damage. Male Sprague-Dawley rats were infused continuously with angiotensin II (350 ng/kg/min) for 8 days. Mean arterial pressure (n = 5), cortical (n = 6) and medullary (n = 7) oxygenation (pO2) were continuously recorded by telemetry and renal tissue injury was scored. Angiotensin II increased arterial pressure gradually to 150 ± 18 mmHg. This was associated with transient reduction of oxygen levels in renal cortex (by 18 ± 2%) and medulla (by 17 ± 6%) at 10 ± 2 and 6 ± 1 hours, respectively after starting infusion. Thereafter oxygen levels normalised to pre-infusion levels and were maintained during the remainder of the infusion period. In rats receiving angiotensin II, adding losartan to drinking water (300 mg/L) only induced transient increase in renal oxygenation, despite normalisation of arterial pressure. In rats, renal hypoxia is only a transient phenomenon during initiation of angiotensin II-induced hypertension.

Nitric Oxide Synthase Inhibition Induces Renal Medullary Hypoxia in Conscious Rats.

Background Renal hypoxia, implicated as crucial factor in onset and progression of chronic kidney disease, may be attributed to reduced nitric oxide because nitric oxide dilates vasculature and inhibits mitochondrial oxygen consumption. We hypothesized that chronic nitric oxide synthase inhibition would induce renal hypoxia. Methods and Results Oxygen-sensitive electrodes, attached to telemeters, were implanted in either renal cortex (n=6) or medulla (n=7) in rats. After recovery and stabilization, baseline oxygenation ( pO 2) was recorded for 1 week. To inhibit nitric oxide synthase, N-ω-nitro-l-arginine (L-NNA; 40 mg/kg/day) was administered via drinking water for 2 weeks. A separate group (n=8), instrumented with blood pressure telemeters, followed the same protocol. L-NNA rapidly induced hypertension (165±6 versus 108±3 mm Hg; P<0.001) and proteinuria (79±12 versus 17±2 mg/day; P<0.001). Cortical pO 2, after initially dipping, returned to baseline and then increased. Medullary pO 2 decreased progressively (up to -19±6% versus baseline; P<0.05). After 14 days of L-NNA, amplitude of diurnal medullary pO 2 was decreased (3.7 [2.2-5.3] versus 7.9 [7.5-8.4]; P<0.01), whereas amplitudes of blood pressure and cortical pO 2 were unaltered. Terminal glomerular filtration rate (1374±74 versus 2098±122 µL/min), renal blood flow (5014±336 versus 9966±905 µL/min), and sodium reabsorption efficiency (13.0±0.8 versus 22.8±1.7 µmol/µmol) decreased (all P<0.001). Conclusions For the first time, we show temporal development of renal cortical and medullary oxygenation during chronic nitric oxide synthase inhibition in unrestrained conscious rats. Whereas cortical pO 2 shows transient changes, medullary pO 2 decreased progressively. Chronic L-NNA leads to decreased renal perfusion and sodium reabsorption efficiency, resulting in progressive medullary hypoxia, suggesting that juxtamedullary nephrons are potentially vulnerable to prolonged nitric oxide depletion.

Deletion of endothelial arginase 1 does not improve vasomotor function in diabetic mice.

Endothelial arginase 1 was ablated to assess whether this prevents hyperglycemia-induced endothelial dysfunction by improving arginine availability for nitric oxide production. Endothelial Arg1-deficient mice (Arg1-KOTie2 ) were generated by crossing Arg1fl/fl (controls) with Tie2Cretg/- mice and analyzed by immunohistochemistry, measurements of hemodynamics, and wire myography. Ablation was confirmed by immunohistochemistry. Mean arterial blood pressure was similar in conscious male control and Arg1-KOTie2 mice. Depletion of circulating arginine by intravenous infusion of arginase 1 or inhibition of nitric oxide synthase activity with L-NG -nitro-arginine methyl ester increased mean arterial pressure similarly in control (9 ± 2 and 34 ± 2 mmHg, respectively) and Arg1-KOTie2 mice (11 ± 3 and 38 ± 4 mmHg, respectively). Vasomotor responses were studied in isolated saphenous arteries of 12- and 34-week-old Arg1-KOTie2 and control animals by wire myography. Diabetes was induced in 10-week-old control and Arg1-KOTie2 mice with streptozotocin, and vasomotor responses were studied 10 weeks later. Optimal arterial diameter, contractile responses to phenylephrine, and relaxing responses to acetylcholine and sodium nitroprusside were similar in normoglycemic control and Arg1-KOTie2 mice. The relaxing response to acetylcholine was dependent on the availability of extracellular l-arginine. In the diabetic mice, arterial relaxation responses to endothelium-dependent hyperpolarization and to exogenous nitric oxide were impaired. The data show that endothelial ablation of arginase 1 in mice does not markedly modify smooth muscle and endothelial functions of a resistance artery under normo- and hyperglycemic conditions.

Testing the effectivity of the mixed virtual reality training Into D'mentia for informal caregivers of people with dementia: protocol for a longitudinal, quasi-experimental study.

INTRODUCTION: Informal caregivers for people with dementia (hereafter: caregivers) often feel (over)burdened by the care for a loved one with dementia, and this can have various deleterious effects on both caregivers and patients. Support for caregivers is urgently needed, and for this reason, a dementia simulator (Into D'mentia) was developed in which caregivers experience what it is like to have dementia. The simulator attempts to heighten caregivers' empathy and understanding for the patient and, in turn, diminish their own caregiver burden. The current study evaluates whether the simulator is effective on a number of outcomes. METHODS AND ANALYSIS: A longitudinal, quasi-experimental study is ongoing in the Netherlands. We aim to recruit 142 caregivers in total divided over two groups: 71 caregivers in the intervention group and 71 caregivers in the control group. All participants will complete interviews and questionnaires at four time points: at baseline, 1 week, 2.5 months and 15 months after the training. The primary outcomes include empathy, caregiver burden, caregiver's sense of competence, social reliance, anxiety, depression and caregivers' subjective and objective health. ETHICS AND DISSEMINATION: This study is being carried out in agreement with the Declaration of Helsinki, and the protocol has been approved by the local ethics committees. REGISTRATION DETAILS: This study is registered with The Netherlands National Trial Register (NNTR5856).

Circadian Rhythm in Kidney Tissue Oxygenation in the Rat.

Blood pressure, renal hemodynamics, electrolyte, and water excretion all display diurnal oscillation. Disturbance of these patterns is associated with hypertension and chronic kidney disease. Kidney oxygenation is dependent on oxygen delivery and consumption that in turn are determined by renal hemodynamics and metabolism. We hypothesized that kidney oxygenation also demonstrates 24-h periodicity. Telemetric oxygen-sensitive carbon paste electrodes were implanted in Sprague-Dawley rats (250-300 g), either in renal medulla (n = 9) or cortex (n = 7). Arterial pressure (MAP) and heart rate (HR) were monitored by telemetry in a separate group (n = 8). Data from 5 consecutive days were analyzed for rhythmicity by cosinor analysis. Diurnal electrolyte excretion was assessed by metabolic cages. During lights-off, oxygen levels increased to 105.3 ± 2.1% in cortex and 105.2 ± 3.8% in medulla. MAP was 97.3 ± 1.5 mmHg and HR was 394.0 ± 7.9 bpm during lights-off phase and 93.5 ± 1.3 mmHg and 327.8 ± 8.9 bpm during lights-on. During lights-on, oxygen levels decreased to 94.6 ± 1.4% in cortex and 94.2 ± 8.5% in medulla. There was significant 24-h periodicity in cortex and medulla oxygenation. Potassium excretion (1,737 ± 779 vs. 895 ± 132 µmol/12 h, P = 0.005) and the distal Na+/K+ exchange (0.72 ± 0.02 vs. 0.59 ± 0.02 P < 0.001) were highest in the lights-off phase, this phase difference was not found for sodium excretion (P = 0.4). It seems that oxygen levels in the kidneys follow the pattern of oxygen delivery, which is known to be determined by renal blood flow and peaks in the active phase (lights-off).

Dual NEP/ECE inhibition improves endothelial function in mesenteric resistance arteries of 32-week-old SHR.

Endothelin 1 (ET-1), a potent vasoconstrictor, pro-mitogenic and pro-inflammatory peptide, may promote development of endothelial dysfunction and arterial remodeling. ET-1 can be formed through cleavage of big-ET-1 by endothelin-converting enzyme (ECE) or neutral endopeptidase (NEP). We investigated whether chronic treatment with the novel dual NEP/ECE inhibitor SOL1 improves functional and structural properties of resistance-sized arteries of 32-week-old male spontaneously hypertensive rats (SHR). SHR received a chronic 4-week treatment with SOL1, losartan or hydralazine. We then compared effects of inhibition of NO synthase (NOS) (100 µM l-NAME), blockade of ETA- and ETB-receptors (10 µM bosentan) and stimulation of the endothelium with 0.001-10 µM acetylcholine (ACh) in isolated third-order mesenteric resistance arteries. Losartan and hydralazine significantly lowered blood pressure. Losartan decreased the media-to-lumen ratio of resistance arteries. l-NAME (1) increased arterial contractile responses to K+ (5.9-40 mM) in the losartan, SOL1 and vehicle group and (2) increased the sensitivity to phenylephrine (PHE; 0.16-20 µM) in the SOL1 group but not in the losartan, hydralazine and vehicle group. Relaxing responses to ACh in the absence or presence of l-NAME during contractions induced by either 10 µM PHE or 40 mM K+ were not altered by any in vivo treatment. Acute treatment with bosentan did, however, significantly improve maximal relaxing responses involving endothelium-derived nitric oxide and -hyperpolarizing factors in the SOL1 group but not in the losartan, hydralazine or vehicle group. Thus, chronic inhibition of NEP/ECE improved basal endothelial function but did not alter blood pressure, resistance artery structure and stimulated endothelium-dependent relaxing responses in 32-week-old SHR.

Exogenous and endogenous angiotensin-II decrease renal cortical oxygen tension in conscious rats by limiting renal blood flow.

KEY POINTS: Our understanding of the mechanisms underlying the role of hypoxia in the initiation and progression of renal disease remains rudimentary. We have developed a method that allows wireless measurement of renal tissue oxygen tension in unrestrained rats. This method provides stable and continuous measurements of cortical tissue oxygen tension (PO2) for more than 2 weeks and can reproducibly detect acute changes in cortical oxygenation. Exogenous angiotensin-II reduced renal cortical tissue PO2 more than equi-pressor doses of phenylephrine, probably because it reduced renal oxygen delivery more than did phenylephrine. Activation of the endogenous renin-angiotensin system in transgenic Cyp1a1Ren2 rats reduced cortical tissue PO2; in this model renal hypoxia precedes the development of structural pathology and can be reversed acutely by an angiotensin-II receptor type 1 antagonist. Angiotensin-II promotes renal hypoxia, which may in turn contribute to its pathological effects during development of chronic kidney disease. ABSTRACT: We hypothesised that both exogenous and endogenous angiotensin-II (AngII) can decrease the partial pressure of oxygen (PO2) in the renal cortex of unrestrained rats, which might in turn contribute to the progression of chronic kidney disease. Rats were instrumented with telemeters equipped with a carbon paste electrode for continuous measurement of renal cortical tissue PO2. The method reproducibly detected acute changes in cortical oxygenation induced by systemic hyperoxia and hypoxia. In conscious rats, renal cortical PO2 was dose-dependently reduced by intravenous AngII. Reductions in PO2 were significantly greater than those induced by equi-pressor doses of phenylephrine. In anaesthetised rats, renal oxygen consumption was not affected, and filtration fraction was increased only in the AngII infused animals. Oxygen delivery decreased by 50% after infusion of AngII and renal blood flow (RBF) fell by 3.3 ml min-1 . Equi-pressor infusion of phenylephrine did not significantly reduce RBF or renal oxygen delivery. Activation of the endogenous renin-angiotensin system in Cyp1a1Ren2 transgenic rats reduced cortical tissue PO2. This could be reversed within minutes by pharmacological angiotensin-II receptor type 1 (AT1 R) blockade. Thus AngII is an important modulator of renal cortical oxygenation via AT1 receptors. AngII had a greater influence on cortical oxygenation than did phenylephrine. This phenomenon appears to be attributable to the profound impact of AngII on renal oxygen delivery. We conclude that the ability of AngII to promote renal cortical hypoxia may contribute to its influence on initiation and progression of chronic kidney disease.

Quantification of Endothelial αvß3 Expression with High-Frequency Ultrasound and Targeted Microbubbles: In Vitro and In Vivo Studies.

Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques. The precursory molecular or cellular pro-angiogenic events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. This study was designed to investigate the feasibility of ultrasound molecular imaging of endothelial αvß3 expression in vitro and in vivo using αvß3-targeted ultrasound contrast agents (UCAs). In the in vitro study, αvß3 expression was confirmed by immunofluorescence in a murine endothelial cell line and detected using the targeted UCA and ultrasound imaging at 18-MHz transmit frequency. In the in vivo study, expression of endothelial αvß3 integrin in murine carotid artery vessels and microvessels of the salivary gland was quantified using targeted UCA and high-frequency ultrasound in seven animals. Our results indicated that endothelial αvß3 expression was significantly higher in the carotid arterial wall containing atherosclerotic lesions than in arterial segments without any lesions. We also found that the salivary gland can be used as an internal positive control for successful binding of targeted UCA to αvß3 integrin. In conclusion, αvß3-targeted UCA allows non-invasive assessment of the expression levels of αvß3 on the vascular endothelium and may provide potential insights into early atherosclerotic plaque detection and treatment monitoring.

The effect of prolonged dietary nitrate supplementation on atherosclerosis development.

BACKGROUND: Short term dietary nitrate or nitrite supplementation has nitric oxide (NO)-mediated beneficial effects on blood pressure and inflammation and reduces mitochondrial oxygen consumption, possibly preventing hypoxia. As these processes are implicated in atherogenesis, dietary nitrate was hypothesized to prevent plaque initiation, hypoxia and inflammation. AIMS: Study prolonged nitrate supplementation on atherogenesis, hypoxia and inflammation in low density lipoprotein receptor knockout mice (LDLr(-/-)). METHODS: LDLr(-/-) mice were administered sodium-nitrate or equimolar sodium-chloride in drinking water alongside a western-type diet for 14 weeks to induce atherosclerosis. Plasma nitrate, nitrite and hemoglobin-bound nitric oxide were measured by chemiluminescence and electron parametric resonance, respectively. RESULTS: Plasma nitrate levels were elevated after 14 weeks of nitrate supplementation (NaCl: 40.29 ± 2.985, NaNO3: 78.19 ± 6.837, p < 0.0001). However, prolonged dietary nitrate did not affect systemic inflammation, hematopoiesis, erythropoiesis and plasma cholesterol levels, suggesting no severe side effects. Surprisingly, neither blood pressure, nor atherogenesis were altered. Mechanistically, plasma nitrate and nitrite were elevated after two weeks (NaCl: 1.0 ± 0.2114, NaNO3: 3.977 ± 0.7371, p < 0.0001), but decreased over time (6, 10 and 14 weeks). Plasma nitrite levels even reached baseline levels at 14 weeks (NaCl: 0.7188 ± 0.1072, NaNO3: 0.9723 ± 0.1279 p = 0.12). Also hemoglobin-bound NO levels were unaltered after 14 weeks. This compensation was not due to altered eNOS activity or conversion into peroxynitrite and other RNI, suggesting reduced nitrite formation or enhanced nitrate/nitrite clearance. CONCLUSION: Prolonged dietary nitrate supplementation resulted in compensation of nitrite and NO levels and did not affect atherogenesis or exert systemic side effects.

Delayed Intervention With Pyridoxamine Improves Metabolic Function and Prevents Adipose Tissue Inflammation and Insulin Resistance in High-Fat Diet-Induced Obese Mice.

Obesity is associated with an increased risk for the development of type 2 diabetes and vascular complications. Advanced glycation end products are increased in adipose tissue and have been associated with insulin resistance, vascular dysfunction, and inflammation of adipose tissue. Here, we report that delayed intervention with pyridoxamine (PM), a vitamin B6 analog that has been identified as an antiglycating agent, protected against high-fat diet (HFD)-induced body weight gain, hyperglycemia, and hypercholesterolemia, compared with mice that were not treated. In both HFD-induced and db/db obese mice, impaired glucose metabolism and insulin resistance were prevented by PM supplementation. PM inhibited the expansion of adipose tissue and adipocyte hypertrophy in mice. In addition, adipogenesis of murine 3T3-L1 and human Simpson-Golabi-Behmel Syndrome preadipocytes was dose- and time-dependently reduced by PM, as demonstrated by Oil Red O staining and reduced expression of adipogenic differentiation genes. No ectopic fat deposition was found in the liver of HFD mice. The high expression of proinflammatory genes in visceral adipose tissue of the HFD group was significantly attenuated by PM. Treatment with PM partially prevented HFD-induced mild vascular dysfunction. Altogether, these findings highlight the potential of PM to serve as an intervention strategy in obesity.

Quantification of bound microbubbles in ultrasound molecular imaging.

Molecular markers associated with diseases can be visualized and quantified noninvasively with targeted ultrasound contrast agent (t-UCA) consisting of microbubbles (MBs) that can bind to specific molecular targets. Techniques used for quantifying t-UCA assume that all unbound MBs are taken out of the blood pool few minutes after injection and only MBs bound to the molecular markers remain. However, differences in physiology, diseases, and experimental conditions can increase the longevity of unbound MBs. In such conditions, unbound MBs will falsely be quantified as bound MBs. We have developed a novel technique to distinguish and classify bound from unbound MBs. In the post-processing steps, first, tissue motion was compensated using block-matching (BM) techniques. To preserve only stationary contrast signals, a minimum intensity projection (MinIP) or 20th-percentile intensity projection (PerIP) was applied. The after-flash MinIP or PerIP was subtracted from the before-flash MinIP or PerIP. In this way, tissue artifacts in contrast images were suppressed. In the next step, bound MB candidates were detected. Finally, detected objects were tracked to classify the candidates as unbound or bound MBs based on their displacement. This technique was validated in vitro, followed by two in vivo experiments in mice. Tumors (n = 2) and salivary glands of hypercholesterolemic mice (n = 8) were imaged using a commercially available scanner. Boluses of 100 µL of a commercially available t-UCA targeted to angiogenesis markers and untargeted control UCA were injected separately. Our results show considerable reduction in misclassification of unbound MBs as bound ones. Using our method, the ratio of bound MBs in salivary gland for images with targeted UCA versus control UCA was improved by up to two times compared with unprocessed images.

Echogenic perfluorohexane-loaded macrophages adhere in vivo to activated vascular endothelium in mice, an explorative study.

BACKGROUND: Macrophages may concentrate ultrasound contrast agents and exhibit selective adhesion to activated endothelium. The present study investigates in mice the potential of perfluorohexane (PFH) loaded macrophages to act as ultrasound contrast agent with high reflectivity and specifically targeted at (atherosclerotic) vascular lesions. METHODS: Lung passage was evaluated with a mouse echo scanner after injection, at a slow pace or as a bolus, of varying doses of PFH-loaded and unloaded bone marrow macrophages (BMM) into the jugular vein. The interaction of PFH-loaded and unloaded BMM with TNF-α stimulated carotid artery endothelium after tail vein injection was assessed by means of intravital microscopy. RESULTS: High doses of jugular vein injected PFH-loaded BMM were visible with ultrasound in the pulmonary artery and detectable in the carotid artery. At intravital microscopy, tail vein injected BMM exhibited rolling and adhesion behavior at the TNF-α stimulated carotid endothelium, similar to that of native blood leukocytes. Rolling behavior was not different between PFH-loaded and unloaded BMM (p = 0.38). CONCLUSION: In vivo, perfluorohexane loaded macrophages pass the pulmonary circulation and appear on the arterial side. Moreover, they roll and adhere selectively to activated endothelium under physiological flow conditions. These findings indicate that perfluorohexane loaded BMM could be used to study processes in vivo where endothelial activation plays a role, such as atherosclerosis.

Reversal of hypoxia in murine atherosclerosis prevents necrotic core expansion by enhancing efferocytosis.

OBJECTIVE: Advanced murine and human plaques are hypoxic, but it remains unclear whether plaque hypoxia is causally related to atherogenesis. Here, we test the hypothesis that reversal of hypoxia in atherosclerotic plaques by breathing hyperoxic carbogen gas will prevent atherosclerosis. APPROACH AND RESULTS: Low-density lipoprotein receptor-deficient mice (LDLR(-/-)) were fed a Western-type diet, exposed to carbogen (95% O2, 5% CO2) or air, and the effect on plaque hypoxia, size, and phenotype was studied. First, the hypoxic marker pimonidazole was detected in murine LDLR(-/-) plaque macrophages from plaque initiation onwards. Second, the efficacy of breathing carbogen (90 minutes, single exposure) was studied. Compared with air, carbogen increased arterial blood pO2 5-fold in LDLR(-/-) mice and reduced plaque hypoxia in advanced plaques of the aortic root (-32%) and arch (-84%). Finally, the effect of repeated carbogen exposure on progression of atherosclerosis was studied in LDLR(-/-) mice fed a Western-type diet for an initial 4 weeks, followed by 4 weeks of diet and carbogen or air (both 90 min/d). Carbogen reduced plaque hypoxia (-40%), necrotic core size (-37%), and TUNEL(+) (terminal uridine nick-end labeling positive) apoptotic cell content (-50%) and increased efferocytosis of apoptotic cells by cluster of differentiation 107b(+) (CD107b, MAC3) macrophages (+36%) in advanced plaques of the aortic root. Plaque size, plasma cholesterol, hematopoiesis, and systemic inflammation were unchanged. In vitro, hypoxia hampered efferocytosis by bone marrow-derived macrophages, which was dependent on the receptor Mer tyrosine kinase. CONCLUSIONS: Carbogen restored murine plaque oxygenation and prevented necrotic core expansion by enhancing efferocytosis, likely via Mer tyrosine kinase. Thus, plaque hypoxia is causally related to necrotic core expansion.