Magnetic resonance imaging contrast-enhancement with superparamagnetic iron oxide nanoparticles amplifies macrophage foam cell apoptosis in human and murine atherosclerosis.

AIMS: (Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidaemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidaemic models of atherosclerosis. METHODS AND RESULTS: RAW264.7 foam cells, exposed in vitro to ferumoxide (dextran-coated SPIO), ferumoxtran (dextran-coated USPIO), or ferumoxytol [carboxymethyl (CM) dextran-coated USPIO] (all 1 mg Fe/mL) showed increased apoptosis and reactive oxygen species accumulation for ferumoxide and ferumoxtran, whereas ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of ferumoxide (0.3 mg Fe/kg) and ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidaemic ApoE-/- (n = 9/group) and LDLR-/- (n = 9-16/group) mice that had received single IV injections compared with saline-treated controls. Again, ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (-68%, P < 0.05) and in plaques from LDLR-/- mice (-60%, P < 0.001, n = 8/group). Repeated ferumoxtran injections of LDLR-/- mice with pre-existing atherosclerosis enhanced plaque inflammation and apoptosis but did not alter plaque size. Strikingly, carotid artery plaques of endarterectomy patients who received ferumoxtran (2.6 mg Fe/kg) before surgery (n = 9) also showed five-fold increased apoptosis (18.2 vs. 3.7%, respectively; P = 0.004) compared with controls who did not receive ferumoxtran. Mechanistically, neither coating nor particle size seemed accountable for the observed cytotoxicity of ferumoxide and ferumoxtran. CONCLUSIONS: Ferumoxide and ferumoxtran, but not ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with advanced atherosclerosis.

Outcomes-Based Selection Into Medical School: Predicting Excellence in Multiple Competencies During the Clinical Years.

PURPOSE: Medical school selection committees aim to identify the best possible students and, ultimately, the best future doctors from a large, well-qualified, generally homogeneous pool of applicants. Constructive alignment of medical school selection, curricula, and assessment with the ultimate outcomes (e.g., CanMEDS roles) has been proposed as means to attain this goal. Whether this approach is effective has not yet been established. The authors addressed this gap by assessing the relationship between performance in an outcomes-based selection procedure and performance during the clinical years of medical school. METHOD: Two groups of students were compared: (1) those admitted into Maastricht University Medical School via an outcomes-based selection procedure and (2) those rejected through this procedure who were admitted into the program through a national, grade-point-average-based lottery. The authors compared performance scores of students from the 2 groups on all 7 CanMEDS roles, using assessment data gathered during clinical rotations. The authors examined data from 3 cohorts (2011-2013). RESULTS: Students admitted through the local, outcomes-based selection procedure significantly outperformed the initially rejected but lottery-admitted students in all years, and the differences between groups increased over time. The selected students performed significantly better in the CanMEDS roles of Communicator, Collaborator, and Professional in the first year of clinical rotations; in these 3 roles-plus Organizer-in the second year; and in 2 additional roles (Advocate and Scholar-all except Medical Expert) at the end of their clinical training. CONCLUSIONS: A constructively aligned selection procedure has increasing predictive value across the clinical years of medical school compared with a GPA-based lottery procedure. The data reported here suggest that constructive alignment of selection, curricula, and assessment to ultimate outcomes is effective in creating a selection procedure predictive of clinical performance.

Opening the black box of selection.

Medical school selection is currently in the paradoxical situation in which selection tools may predict study outcomes, but which constructs are actually doing the predicting is unknown (the 'black box of selection'). Therefore, our research focused on those constructs, answering the question: do the internal structures of the tests in an outcome-based selection procedure reflect the content that was intended to be measured? Downing's validity framework was applied to organize evidence for construct validity, focusing on evidence related to content and internal structure. The applied selection procedure was a multi-tool, CanMEDS-based procedure comprised of a video-based situational judgement test (focused on (inter)personal competencies), and a written aptitude test (reflecting a broader array of CanMEDS competencies). First, we examined content-related evidence pertaining to the creation and application of the competency-based selection blueprint and found that the set-up of the selection procedure was a robust, transparent and replicable process. Second, the internal structure of the selection tests was investigated by connecting applicants' performance on the selection tests to the predetermined blueprint using cognitive diagnostic modeling. The data indicate 89% overlap between the expected and measured constructs. Our results support the notion that the focus placed on creating the right content and following a competency-blueprint was effective in terms of internal structure: most items measured what they were intended to measure. This way of linking a predetermined blueprint to the applicants' results sheds light into the 'black box of selection' and can be used to support the construct validity of selection procedures.

Does selection pay off? A cost-benefit comparison of medical school selection and lottery systems.

CONTEXT: Resources for medical education are becoming more constrained, whereas accountability in medical education is increasing. In this constrictive environment, medical schools need to consider and justify their selection procedures in terms of costs and benefits. To date, there have been no studies focusing on this aspect of selection. OBJECTIVES: We aimed to examine and compare the costs and benefits of two different approaches to admission into medical school: a tailored, multimethod selection process versus a lottery procedure. Our goal was to assess the relative effectiveness of each approach and to compare these in terms of benefits and costs from the perspective of the medical school. METHODS: The study was conducted at Maastricht University Medical School, at which the selection process and a weighted lottery procedure ran in parallel for 3 years (2011-2013). The costs and benefits of the selection process were compared with those of the lottery procedure over three student cohorts throughout the Bachelor's programme. The extra costs of selection represented the monetary investment of the medical school in conducting the selection procedure; the benefits were derived from the increase in income generated by the prevention of dropout and the reductions in extra costs facilitated by decreases in the repetition of blocks and objective structured clinical examinations. RESULTS: The tailor-made selection procedure cost about €139 000 when extrapolated to a full cohort of students (n = 286). The lottery procedure came with negligible costs for the medical school. However, the average benefits of selection compared with the lottery system added up to almost €207 000. CONCLUSIONS: This study not only shows that conducting a cost-benefit comparison is feasible in the context of selection for medical school, but also that an 'expensive' selection process can be cost-beneficial in comparison with an 'inexpensive' lottery system. We encourage other medical schools to examine the cost-effectiveness of their own selection processes in relation to student outcomes in order to extend knowledge on this important topic.

Selection into medicine: the predictive validity of an outcome-based procedure.

BACKGROUND: Medical schools must select students from a large pool of well-qualified applicants. A challenging issue set forward in the broader literature is that of which cognitive and (inter)personal qualities should be measured to predict diverse later performance. To address this gap, we designed a 'backward chaining' approach to selection, based on the competences of a 'good doctor'. Our aim was to examine if this outcome-based selection procedure was predictive of study success in a medical bachelor program. METHODS: We designed a multi-tool selection procedure, blueprinted to the CanMEDS competency framework. The relationship between performance at selection and later study success across a three-year bachelor program was examined in three cohorts. Study results were compared between selection-positive and selection-negative (i.e. primarily rejected) students. RESULTS: Selection-positive students outperformed their selection-negative counterparts throughout the entire bachelor program on assessments measuring cognitive (e.g. written exams), (inter)personal and combined outcomes (i.e. OSCEs). Of the 30 outcome variables, selection-positive students scored significantly higher in 11 cases. Fifteen other, non-significant between-group differences were also in favor of the selection-positives. An overall comparison using a sign test indicated a significant difference between both groups (p < 0.001), despite equal pre-university GPAs. CONCLUSIONS: The use of an outcome-based selection approach seems to address some of the predictive validity limitations of commonly-used selection tools. Selection-positive students significantly outperformed their selection-negative counterparts across a range of cognitive, (inter)personal, and mixed outcomes throughout the entire three-year bachelor in medicine.

Cathepsin K Deficiency Prevents the Aggravated Vascular Remodeling Response to Flow Cessation in ApoE-/- Mice.

Cathepsin K (catK) is a potent lysosomal cysteine protease involved in extracellular matrix (ECM) degradation and inflammatory remodeling responses. Here we have investigated the contribution of catK deficiency on carotid arterial remodeling in response to flow cessation in apoE-/- and wild type (wt) background. Ligation-induced hyperplasia is considerably aggravated in apoE-/- versus wt mice. CatK protein expression was significantly increased in neointimal lesions of apoE-/- compared with wt mice, suggesting a role for catK in intimal hyperplasia under hyperlipidemic conditions. Surprisingly, CatK deficiency completely blunted the augmented hyperplastic response to flow cessation in apoE-/-, whereas vascular remodeling in wt mice was unaffected. As catK deficiency did neither alter lesion collagen content and elastic laminae fragmentation in vivo, we focused on effects of catK on (systemic) inflammatory responses. CatK deficiency significantly reduced circulating CD3 T-cell numbers, but increased the regulatory T cell subset in apoE-/- but not wt mice. Moreover, catK deficiency changed CD11b+Ly6G-Ly6C high monocyte distribution in apoE-/- but not wt mice and tended to favour macrophage M2a polarization. In conclusion, catK deficiency almost completely blunted the increased vascular remodeling response of apoE-/- mice to flow cessation, possibly by correcting hyperlipidemia-associated pro-inflammatory effects on the peripheral immune response.

High-sensitive troponin T assay for the diagnosis of acute myocardial infarction: an economic evaluation.

BACKGROUND: Delayed diagnosis and treatment of Acute Myocardial Infarction (AMI) has a major adverse impact on prognosis in terms of both morbidity and mortality. Since conventional cardiac Troponin assays have a low sensitivity for diagnosing AMI in the first hours after myocardial necrosis, high-sensitive assays have been developed. The aim of this study was to assess the cost effectiveness of a high-sensitive Troponin T assay (hsTnT), alone or combined with the heart-type fatty acid-binding protein (H-FABP) assay in comparison with the conventional cardiac Troponin (cTnT) assay for the diagnosis of AMI in patients presenting to the hospital with chest pain. METHODS: We performed a cost-utility analysis (quality adjusted life years-QALYs) and a cost effectiveness analysis (life years gained-LYGs) based on a decision analytic model, using a health care perspective in the Dutch context and a life time time-horizon. The robustness of model predictions was explored using one-way and probabilistic sensitivity analyses. RESULTS: For a life time incremental cost of 30.70 Euros, use of hsTnT over conventional cTnT results in gain of 0.006 Life Years and 0.004 QALY. It should be noted here that hsTnT is a diagnostic intervention which costs only 4.39 Euros/test more than the cTnT test. The ICER generated with the use of hsTnT based diagnostic strategy comparing with the use of a cTnT-based strategy, is 4945 Euros per LYG and 7370 Euros per QALY. The hsTnT strategy has the highest probability of being cost effective at thresholds between 8000 and 20000 Euros per QALY. The combination of hsTnT and h-FABP strategy's probability of being cost effective remains lower than hsTnT at all willingness to pay thresholds. CONCLUSION: Our analysis suggests that hsTnT assay is a very cost effective diagnostic tool relative to conventional TnT assay. Combination of hsTnT and H-FABP does not offer any additional economic and health benefit over hsTnT test alone.

Chemokines CCL3/MIP1α, CCL5/RANTES and CCL18/PARC are independent risk predictors of short-term mortality in patients with acute coronary syndromes.

Cytokines play an important role in ischemic injury and repair. However, little is known about their prognostic value in cardiovascular disease. The aim of this study was to investigate the prognostic importance of chemokines CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC for the risk of future cardiovascular events in patients with acute coronary syndromes (ACS). Baseline levels of CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC were determined in ACS patients from the Bad Nauheim ACS II registry (n = 609). During the following 200 days, patients were monitored for the occurrence of fatal and non-fatal cardiovascular events. Patients with CCL3/MIP1α, CCL5/RANTES and CCL18/PARC concentrations in the highest tertile were associated with an increased risk of a fatal event during follow-up (HR: 2.19, 95%CI: 1.04-4.61 for CCL3/MIP1α, HR: 3.45, 95%CI: 1.54-7.72 for CCL5/RANTES and HR: 3.14, 95%CI: 1.33-7.46 for CCL18/PARC). This risk was highest for patients with all three biomarkers concentrations in the upper tertile (HR: 2.52, 95%CI: 1.11-5.65). Together with known risk predictors of cardiovascular events, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC combined improved the c-statistics from 0.74 to 0.81 (p = 0.007). In conclusion, CCL3/MIP-1α, CCL5/RANTES and CCL18/PARC are independently associated with the risk of short-term mortality in ACS patients. Combining all three biomarkers further increased their prognostic value.

Cathepsin K gene disruption does not affect murine aneurysm formation.

Cathepsin K (catK), a lysosomal cysteine protease, exerts strong elastinolytic and collagenolytic activity and is implicated in a range of pathological disorders including cardiovascular disease. CatK expression was found to be elevated in human aortic aneurysm pointing to a role in this vasculopathy. In the angiotensin II (Ang II)-induced mouse model for aneurysm formation, catK, S and C expression was strongly upregulated. Therefore, we investigated the effect of catK deficiency on Ang II-induced aneurysm formation in the abdominal aorta of apoE-/- mice. Contrary to our expectations, catK deficiency did not protect against aneurysm formation, nor did it affect medial elastin breaks. Proteolytic activity in abdominal aortic lysates were comparable between apoE-/- and catK-//-apoE-/- mice. Adventitial presence of catS- and catC-expressing cells was significantly increased in catK-/-//apoE-/- versus apoE-/- mice, which might have compensated for the deficiency of catK-derived proteolysis in the aneurysm tissue of catK deficient apoE-/- mice. Circulating granulocytes and activated T cell numbers were significantly increased in Ang II-infused catK-/-//apoE-/- mice, which is consistent with the borderline significant increase in adventitial leukocyte content in catK-/-//apoE-/- compared to apoE-/- mice. Strikingly, despite unchanged proteolytic activity in AAA lesions, collagen content in the aneurysm was significantly increased in catK-//-apoE-/- mice. In conclusion, while catK deficiency has major impact on various vasculopathies, it did not affect murine aneurysm formation.

Noninvasive diagnosis of ruptured peripheral atherosclerotic lesions and myocardial infarction by antibody profiling.

Novel biomarkers, such as circulating (auto)antibody signatures, may improve early detection and treatment of ruptured atherosclerotic lesions and accompanying cardiovascular events, such as myocardial infarction. Using a phage-display library derived from cDNAs preferentially expressed in ruptured peripheral human atherosclerotic plaques, we performed serological antigen selection to isolate displayed cDNA products specifically interacting with antibodies in sera from patients with proven ruptured peripheral atherosclerotic lesions. Two cDNA products were subsequently evaluated on a validation series of patients with peripheral atherosclerotic lesions, healthy controls, and patients with coronary artery disease at different stages. Our biomarker set was able to discriminate between patients with peripheral ruptured lesions and patients with peripheral stable plaques with 100% specificity and 76% sensitivity. Furthermore, 93% of patients with an acute myocardial infarction (AMI) tested positive for our biomarkers, whereas all patients with stable angina pectoris tested negative. Moreover, 90% of AMI patients who initially tested negative for troponin T, for which a positive result is known to indicate myocardial infarction, tested positive for our biomarkers upon hospital admission. In conclusion, antibody profiling constitutes a promising approach for noninvasive diagnosis of atherosclerotic lesions, because a positive serum response against a set of 2 cDNA products showed a strong association with the presence of ruptured peripheral atherosclerotic lesions and myocardial infarction.

Comparison of single-sequence T1w TFE MRI with multisequence MRI for the quantification of lipid-rich necrotic core in atherosclerotic plaque.

PURPOSE: To prospectively determine the accuracy of semiquantitative analysis of the amount of lipid-rich necrotic core (LRNC) in atherosclerotic plaque using multi- as well as single-sequence T1-weighted (w) turbo field echo (TFE) MRI. Histology served as a reference standard. MATERIALS AND METHODS: Sixty-four symptomatic patients with carotid stenosis > or =70% were included and underwent endarterectomy after an MRI scan. Two MRI readers classified relative signal intensities in regions of interest in the vessel wall. The amount of LRNC was determined semiquantitatively using an algorithm based on fixed combinations of multiple MR pulse sequences as well as solely based on T1w TFE images. Interreader agreement was expressed by intraclass correlation coefficients (ICCs). Agreement between MRI and histology was determined by linear regression (R). RESULTS: Interreader reproducibility for quantification of LRNC was high (ICC, 95% confidence interval [CI]): multisequence 0.86 (0.77-0.94), and single sequence 0.91 (0.85-0.95). There was good agreement between MRI and histology for both MR readers for quantification based on multisequence as well as single sequence MRI, 0.80 < or = R < or = 0.85 (P < 0.0001). CONCLUSION: The amount of LRNC using single-sequence T1w TFE MRI is a reproducible, accurate, and fast way to quantify LRNC in carotid atherosclerotic plaque.

Comparison of lipid-rich necrotic core size in symptomatic and asymptomatic carotid atherosclerotic plaque: Initial results.

PURPOSE: To investigate the potential difference in the size of the lipid-rich necrotic core (LRNC) in carotid plaques of symptomatic patients versus asymptomatic patients. Pathological studies established that a large LRNC is an important feature of vulnerable atherosclerotic plaque. Previously, we have demonstrated a high correlation between semiquantitative analysis of the LRNC size in T1-weighted (w) turbo field echo (TFE) MR images and histology. MATERIALS AND METHODS: Thirty-seven patients with carotid stenosis >70% with (n = 26) or without (n = 11) symptoms were included. Three independent MR readers quantified the amount of LRNC with a T1w TFE pulse sequence. The relative amount of LRNC (LRNC score) was defined as sum of cross-sectional area percentages LRNC per carotid plaque. RESULTS: Interreader agreement for the three MR readers was good, with an intraclass correlation coefficient (ICC, 95% confidence interval [CI]) of 0.72 (0.57-0.83). All three MR readers on average found a larger LRNC in the symptomatic group of patients, although this was not statistically significant. The mean LRNC score was 116 +/- 129 and 59 +/- 62 for symptomatic and asymptomatic patients, respectively (P = 0.13). Symptomatic patients showed wide ranges in LRNC scores (0-424), while the range was much lower in the asymptomatic group (0-170). CONCLUSION: Single-sequence T1w TFE may be a promising technique to study atherosclerotic plaque at risk of stroke. Larger studies are warranted to confirm these promising results.

Dead or alive: gene expression profiles of advanced atherosclerotic plaques from autopsy and surgery.

Since inclusion of atherosclerotic tissues from different sources is often indispensable to study the full atherogenic spectrum, we investigated to what extent the expression profiles of advanced, stable atherosclerotic lesions obtained during autopsy and surgery are comparable. The gene expression profiles of human carotids with advanced atherosclerosis obtained at autopsy and at vascular surgery were studied by microarray analysis. Expression analysis was performed both at the single gene (Rosetta, Gene Ontology) and at the pathway level using Ingenuity and Gene Set Enrichment Analysis. In addition, mRNA and protein expression levels were validated using quantitative (q) RT-PCR and immunohistochemistry on unrelated advanced carotid lesions from autopsy and surgery. Microarray analysis indicated that the 97.2% of genes showed similar expression levels in advanced atherosclerotic lesions from autopsy and surgery. While the expression data revealed no differences in common atherosclerotic related pathways such as lipid metabolism and inflammation, the differentially expressed genes were mainly involved in basal cell metabolism and hypoxia driven pathways. qRT-PCR confirmed the differential expression of hypoxia-driven genes VEGF-A (2.3-fold upward arrow), glucose transporter (GLUT)-1 (2.5-fold upward arrow), GLUT3 (8.3-fold upward arrow), and hexokinase 1 (2.4-fold upward arrow) in autopsy vs. surgical specimens. Immunohistochemistry revealed that the transcriptional differences in these hypoxia-related genes were not reflected at the protein level. The gene expression profiles of advanced atherosclerotic lesions from autopsy and surgery are largely similar. However, >500 genes, mostly involved in basal cell metabolism and hypoxia were differentially expressed at mRNA, but not at the protein level.

Cathepsin cysteine proteases in cardiovascular disease.

Extracellular matrix (ECM) remodeling is one of the underlying mechanisms in cardiovascular diseases. Cathepsin cysteine proteases have a central role in ECM remodeling and have been implicated in the development and progression of cardiovascular diseases. Cathepsins also show differential expression in various stages of atherosclerosis, and in vivo knockout studies revealed that deficiency of cathepsin K or S reduces atherosclerosis. Furthermore, cathepsins are involved in lipid metabolism. Cathepsins have the capability to degrade low-density lipoprotein and reduce cholesterol efflux from macrophages, aggravating foam cell formation. Although expression studies also demonstrated differential expression of cathepsins in cardiovascular diseases like aneurysm formation, neointima formation, and neovascularization, in vivo studies to define the exact role of cathepsins in these processes are lacking. Evaluation of the feasibility of cathepsins as a diagnostic tool revealed that serum levels of cathepsins L and S seem to be promising as biomarkers in the diagnosis of atherosclerosis, whereas cathepsin B shows potential as an imaging tool. Furthermore, cathepsin K and S inhibitors showed effectiveness in (pre) clinical evaluation for the treatment of osteoporosis and osteoarthritis, suggesting that cathepsin inhibitors may also have therapeutic effects for the treatment of atherosclerosis.

Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone.

UNLABELLED: Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous bone matrix. INTRODUCTION: The osteoclast resorbs bone by lowering the pH in the resorption lacuna, which is followed by secretion of proteolytic enzymes. One of the enzymes taken to be essential in resorption is the cysteine proteinase, cathepsin K. Some immunolabeling and enzyme inhibitor data, however, suggest that other cysteine proteinases and/or proteolytic enzymes belonging to the group of matrix metalloproteinases (MMPs) may participate in the degradation. In this study, we investigated whether, in addition to cathepsin K, other enzymes participate in osteoclastic bone degradation. MATERIALS AND METHODS: In bones obtained from mice deficient for cathepsin K, B, or L or a combination of K and L, the bone-resorbing activity of osteoclasts was analyzed at the electron microscopic level. In addition, bone explants were cultured in the presence of different selective cysteine proteinase inhibitors and an MMP inhibitor, and the effect on resorption was assessed. Because previous studies showed differences in resorption by calvarial osteoclasts compared with those present in long bones, in all experiments, the two types of bone were compared. Finally, bone extracts were analyzed for the level of activity of cysteine proteinases and the effect of inhibitors hereupon. RESULTS: The analyses of the cathepsin-deficient bone explants showed that, in addition to cathepsin K, calvarial osteoclasts use other cysteine proteinases to degrade bone matrix. It was also shown that, in the absence of cathepsin K, long bone osteoclasts use MMPs for resorption. Cathepsin L proved to be involved in the MMP-mediated resorption of bone by calvarial osteoclasts; in the absence of this cathepsin, calvarial osteoclasts do not use MMPs for resorption. Selective inhibitors of cathepsin K and other cysteine proteinases showed a stronger effect on calvarial resorption than on long bone resorption. CONCLUSIONS: Our findings suggest that (1) cathepsin K-deficient long bone osteoclasts compensate the lack of this enzyme by using MMPs in the resorption of bone matrix; (2) cathepsin L is involved in MMP-mediated resorption by calvarial osteoclasts; (3) in addition to cathepsin K, other, yet unknown, cysteine proteinases are likely to participate in skull bone degradation; and finally, (4) the data provide strong additional support for the existence of functionally different bone-site specific osteoclasts.

Targeted biallelic inactivation of Pten in the mouse prostate leads to prostate cancer accompanied by increased epithelial cell proliferation but not by reduced apoptosis.

The PTEN tumor suppressor gene is frequently inactivated in human tumors, including prostate cancer. Based on the Cre/loxP system, we generated a novel mouse prostate cancer model by targeted inactivation of the Pten gene. In this model, Cre recombinase was expressed under the control of the prostate-specific antigen (PSA) promoter. Conditional biallelic and monoallelic Pten knock-out mice were viable and Pten recombination was prostate-specific. Mouse cohorts were systematically characterized at 4 to 5, 7 to 9, and 10 to 14 months. A slightly increased proliferation rate of epithelial cells was observed in all prostate lobes of monoallelic Pten knock-out mice (PSA-Cre;Pten-loxP/+), but minimal pathologic changes were detected. All homozygous knock-out mice (PSA-Cre;Pten-loxP/loxP) showed an increased size of the luminal epithelial cells, large areas of hyperplasia, focal prostate intraepithelial neoplasia lesions and an increased prostate weight at 4 to 5 months. More extensive prostate intraepithelial neoplasia and focal microinvasion occurred at 7 to 9 months; invasive prostate carcinoma was detected in all male PSA-Cre;Pten-loxP/loxP mice at 10 to 14 months. At 15 to 16 months, a rare lymph node metastasis was found. In hyperplastic cells and in tumor cells, the expression of phospho-AKT was up-regulated. In hyperplastic and tumor cells, expression of luminal epithelial cell cytokeratins was up-regulated; tumor cells were negative for basal epithelial cell cytokeratins. Androgen receptor expression remained detectable at all stages of tumor development. The up-regulation of phospho-AKT correlated with an increased proliferation rate of the epithelial cells, but not with a reduced apoptosis.

Gene profiling in atherosclerosis reveals a key role for small inducible cytokines: validation using a novel monocyte chemoattractant protein monoclonal antibody.

BACKGROUND: Pathological aspects of atherosclerosis are well described, but gene profiles during atherosclerotic plaque progression are largely unidentified. METHODS AND RESULTS: Microarray analysis was performed on mRNA of aortic arches of ApoE-/- mice fed normal chow (NC group) or Western-type diet (WD group) for 3, 4.5, and 6 months. Of 10 176 reporters, 387 were differentially (>2x) expressed in at least 1 group compared with a common reference (ApoE-/-, 3- month NC group). The number of differentially expressed genes increased during plaque progression. Time-related expression clustering and functional grouping of differentially expressed genes suggested important functions for genes involved in inflammation (especially the small inducible cytokines monocyte chemoattractant protein [MCP]-1, MCP-5, macrophage inflammatory protein [MIP]-1alpha, MIP-1beta, MIP-2, and fractalkine) and matrix degradation (cathepsin-S, matrix metalloproteinase-2/12). Validation experiments focused on the gene cluster of small inducible cytokines. Real-time polymerase chain reaction revealed a plaque progression-dependent increase in mRNA levels of MCP-1, MCP-5, MIP-1alpha, and MIP-1beta. ELISA for MCP-1 and MCP-5 showed similar results. Immunohistochemistry for MCP-1, MCP-5, and MIP-1alpha located their expression to plaque macrophages. An inhibiting antibody for MCP-1 and MCP-5 (11K2) was designed and administered to ApoE-/- mice for 12 weeks starting at the age of 5 or 17 weeks. 11K2 treatment reduced plaque area and macrophage and CD45+ cell content and increased collagen content, thereby inducing a stable plaque phenotype. CONCLUSIONS: Gene profiling of atherosclerotic plaque progression in ApoE-/- mice revealed upregulation of the gene cluster of small inducible cytokines. Further expression and in vivo validation studies showed that this gene cluster mediates plaque progression and stability.

Assessment of human atherosclerotic carotid plaque components with multisequence MR imaging: initial experience.

PURPOSE: To prospectively determine, by using a stepwise logistic regression model, the optimal magnetic resonance (MR) weighting (ie, pulse sequence) combinations for plaque assessment and corresponding cutoff values of relative signal intensities (rSIs). MATERIALS AND METHODS: Institutional review board approval and patient consent were obtained. Eleven patients (seven men, four women; mean age +/- standard deviation, 68 years +/- 4) with symptomatic carotid disease and stenosis of more than 70% were investigated at MR imaging before carotid endarterectomy. The MR images were matched with histologic features of the endarterectomy specimens (reference standard). The rSIs (compared with that of muscle tissue) from regions of interest were assessed qualitatively and semiquantitatively. For all major components (calcification, lipid core, intraplaque hemorrhage, and fibrous tissue), optimal cutoff points for the rSIs were determined for five MR weightings by means of receiver operating characteristic curves. The best predicting combinations of these five dichotomized MR weightings were selected by means of stepwise logistic regression analysis. The potential sensitivity and specificity of MR imaging for vulnerable plaque with hemorrhage and/or lipid core were determined. RESULTS: The same optimal MR weighting combinations for identifying the four plaque components were found with qualitative and semiquantitative analysis. Sensitivity and specificity for vulnerable plaque were 93% (95% confidence interval: 77%, 99%) and 96% (95% confidence interval: 86%, 100%), respectively, for the qualitative analysis and 76% (95% confidence interval: 56%, 90%) and 100% (95% confidence interval: 93%, 100%) for the semiquantitative analysis. CONCLUSION: This study demonstrates the potential of a systematic approach of atherosclerotic plaque assessment with multisequence MR imaging by using the information provided from five different MR weightings in a stepwise logistic regression model.