Unravelling the skillset of point-of-care ultrasound: a systematic review.

BACKGROUND: The increasing number of physicians that are trained in point-of-care ultrasound (POCUS) warrants critical evaluation and improvement of current training methods. Performing POCUS is a complex task and it is unknown which (neuro)cognitive mechanisms are most important in competence development of this skill. This systematic review was conducted to identify determinants of POCUS competence development that can be used to optimize POCUS training. METHODS: PubMed, Web of Science, Cochrane Library, Emcare, PsycINFO and ERIC databases were searched for studies measuring ultrasound (US) skills and aptitude. The papers were divided into three categories: "Relevant knowledge", "Psychomotor ability" and 'Visuospatial ability'. The 'Relevant knowledge' category was further subdivided in 'image interpretation', 'technical aspects' and 'general cognitive abilities'. Visuospatial ability was subdivided in visuospatial subcategories based on the Cattell-Horn-Carroll (CHC) Model of Intelligence v2.2, which includes visuospatial manipulation and visuospatial perception. Post-hoc, a meta-analysis was performed to calculate pooled correlations. RESULTS: 26 papers were selected for inclusion in the review. 15 reported on relevant knowledge with a pooled coefficient of determination of 0.26. Four papers reported on psychomotor abilities, one reported a significant relationship with POCUS competence. 13 papers reported on visuospatial abilities, the pooled coefficient of determination was 0.16. CONCLUSION: There was a lot of heterogeneity in methods to assess possible determinants of POCUS competence and POCUS competence acquisition. This makes it difficult to draw strong conclusions on which determinants should be part of a framework to improve POCUS education. However, we identified two determinants of POCUS competence development: relevant knowledge and visuospatial ability. The content of relevant knowledge could not be retrieved in more depth. For visuospatial ability we used the CHC model as theoretical framework to analyze this skill. We could not point out psychomotor ability as a determinant of POCUS competence.

Effect of binocular disparity on learning anatomy with stereoscopic augmented reality visualization: A double center randomized controlled trial.

Binocular disparity provides one of the important depth cues within stereoscopic three-dimensional (3D) visualization technology. However, there is limited research on its effect on learning within a 3D augmented reality (AR) environment. This study evaluated the effect of binocular disparity on the acquisition of anatomical knowledge and perceived cognitive load in relation to visual-spatial abilities. In a double-center randomized controlled trial, first-year (bio)medical undergraduates studied lower extremity anatomy in an interactive 3D AR environment either with a stereoscopic 3D view (n = 32) or monoscopic 3D view (n = 34). Visual-spatial abilities were tested with a mental rotation test. Anatomical knowledge was assessed by a validated 30-item written test and 30-item specimen test. Cognitive load was measured by the NASA-TLX questionnaire. Students in the stereoscopic 3D and monoscopic 3D groups performed equally well in terms of percentage correct answers (written test: 47.9 ± 15.8 vs. 49.1 ± 18.3; P = 0.635; specimen test: 43.0 ± 17.9 vs. 46.3 ± 15.1; P = 0.429), and perceived cognitive load scores (6.2 ± 1.0 vs. 6.2 ± 1.3; P = 0.992). Regardless of intervention, visual-spatial abilities were positively associated with the specimen test scores (η2 = 0.13, P = 0.003), perceived representativeness of the anatomy test questions (P = 0.010) and subjective improvement in anatomy knowledge (P < 0.001). In conclusion, binocular disparity does not improve learning anatomy. Motion parallax should be considered as another important depth cue that contributes to depth perception during learning in a stereoscopic 3D AR environment.

Primary cilia control endothelial permeability by regulating expression and location of junction proteins.

AIMS: Wall shear stress (WSS) determines intracranial aneurysm (IA) development. Polycystic kidney disease (PKD) patients have a high IA incidence and risk of rupture. Dysfunction/absence of primary cilia in PKD endothelial cells (ECs) may impair mechano-transduction of WSS and favour vascular disorders. The molecular links between primary cilia dysfunction and IAs are unknown. METHODS AND RESULTS: Wild-type and primary cilia-deficient Tg737orpk/orpk arterial ECs were submitted to physiological (30 dynes/cm2) or aneurysmal (2 dynes/cm2) WSS, and unbiased transcriptomics were performed. Tg737orpk/orpk ECs displayed a fivefold increase in the number of WSS-responsive genes compared to wild-type cells. Moreover, we observed a lower trans-endothelial resistance and a higher endothelial permeability, which correlated with disorganized intercellular junctions in Tg737orpk/orpk cells. We identified ZO-1 as a central regulator of primary cilia-dependent endothelial junction integrity. Finally, clinical and histological characteristics of IAs from non-PKD and PKD patients were analysed. IAs in PKD patients were more frequently located in the middle cerebral artery (MCA) territory than in non-PKD patients. IA domes from the MCA of PKD patients appeared thinner with less collagen and reduced endothelial ZO-1 compared with IA domes from non-PKD patients. CONCLUSION: Primary cilia dampen the endothelial response to aneurysmal low WSS. In absence of primary cilia, ZO-1 expression levels are reduced, which disorganizes intercellular junctions resulting in increased endothelial permeability. This altered endothelial function may not only contribute to the severity of IA disease observed in PKD patients, but may also serve as a potential diagnostic tool to determine the vulnerability of IAs.

Development of a Virtual Three-Dimensional Assessment Scenario for Anatomical Education.

In anatomical education three-dimensional (3D) visualization technology allows for active and stereoscopic exploration of anatomy and can easily be adopted into medical curricula along with traditional 3D teaching methods. However, most often knowledge is still assessed with two-dimensional (2D) paper-and-pencil tests. To address the growing misalignment between learning and assessment, this viewpoint commentary highlights the development of a virtual 3D assessment scenario and perspectives from students and teachers on the use of this assessment tool: a 10-minute session of anatomical knowledge assessment with real-time interaction between assessor and examinee, both wearing a HoloLens and sharing the same stereoscopic 3D augmented reality model. Additionally, recommendations for future directions, including implementation, validation, logistic challenges, and cost-effectiveness, are provided. Continued collaboration between developers, researchers, teachers, and students is critical to advancing these processes.

Development of a patient-oriented Hololens application to illustrate the function of medication after myocardial infarction.

Aims: Statin treatment is one of the hallmarks of secondary prevention after myocardial infarction. Adherence to statins tends to be difficult and can be improved by patient education. Novel technologies such as mixed reality (MR) expand the possibilities to support this process. To assess if an MR medication-application supports patient education focused on function of statins after myocardial infarction. Methods and results: A human-centred design-approach was used to develop an MR statin tool for Microsoft HoloLens™. Twenty-two myocardial infarction patients were enrolled; 12 tested the application, 10 patients were controls. Clinical, demographic, and qualitative data were obtained. All patients performed a test on statin knowledge. To test if patients with a higher tendency to become involved in virtual environments affected test outcome in the intervention group, validated Presence- and Immersive Tendency Questionnaires (PQ and ITQ) were used. Twenty-two myocardial infarction patients (ST-elevation myocardial infarction, 18/22, 82%) completed the study. Ten out of 12 (83%) patients in the intervention group improved their statin knowledge by using the MR application (median 8 points, IQR 8). Test improvement was mainly the result of increased understanding of statin mechanisms in the body and secondary preventive effects. A high tendency to get involved and focused in virtual environments was moderately positive correlated with better test improvement (r = 0.57, P < 0.05). The median post-test score in the control group was poor (median 6 points, IQR 4). Conclusions: An MR statin education application can be applied effectively in myocardial infarction patients to explain statin function and importance.

Stereoscopic three-dimensional visualisation technology in anatomy learning: A meta-analysis.

OBJECTIVES: The features that contribute to the apparent effectiveness of three-dimensional visualisation technology [3DVT] in teaching anatomy are largely unknown. The aim of this study was to conduct a systematic review and meta-analysis of the role of stereopsis in learning anatomy with 3DVT. METHODS: The review was conducted and reported according to PRISMA Standards. Literature search of English articles was performed using EMBASE, MEDLINE, CINAHL EBSCOhost, ERIC EBSCOhost, Cochrane CENTRAL, Web of Science and Google Scholar databases until November 2019. Study selection, data extraction and study appraisal were performed independently by two authors. Articles were assessed for methodological quality using the Medical Education Research Study Quality Instrument and the Cochrane Collaboration's tool for assessing the risk of bias. For quantitative analysis, studies were grouped based on relative between-intervention differences in instructional methods and type of control conditions. RESULTS: A total of 3934 citations were obtained of which 67 underwent a full-text review. Ultimately, 13 randomised controlled trials were included in the meta-analysis. When interactive, stereoscopic 3D models were compared to interactive, monoscopic 3D models within a single level of instructional design, for example isolating stereopsis as the only true manipulated element in the experimental design, an effect size [ES] of 0.53 (95% confidence interval [CI] 0.26-0.80; P < .00001) was found. In comparison with 2D images within multiple levels of instructional design, an effect size of 0.45 (95% CI 0.10-0.81; P < .002) was found. Stereopsis had no effect on learning when utilised with non-interactive 3D images (ES = -0.87, 95% CI -2.09-0.35; P = .16). CONCLUSION: Stereopsis is an important distinguishing element of 3DVT that has a significant positive effect on acquisition of anatomical knowledge when utilised within an interactive 3D environment. A distinction between stereoscopic and monoscopic 3DVT is essential to make in anatomical education and research.

SERIES: eHealth in primary care. Part 3: eHealth education in primary care.

BACKGROUND: Education is essential to the integration of eHealth into primary care, but eHealth is not yet embedded in medical education. OBJECTIVES: In this opinion article, we aim to support organisers of Continuing Professional Development (CPD) and teachers delivering medical vocational training by providing recommendations for eHealth education. First, we describe what is required to help primary care professionals and trainees learn about eHealth. Second, we elaborate on how eHealth education might be provided. DISCUSSION: We consider four essential topics. First, an understanding of existing evidence-based eHealth applications and conditions for successful development and implementation. Second, required digital competencies of providers and patients. Third, how eHealth changes patient-provider and provider-provider relationships and finally, understanding the handling of digital data. Educational activities to address these topics include eLearning, blended learning, courses, simulation exercises, real-life practice, supervision and reflection, role modelling and community of practice learning. More specifically, a CanMEDS framework aimed at defining curriculum learning goals can support eHealth education by describing roles and required competencies. Alternatively, Kern's conceptual model can be used to design eHealth training programmes that match the educational needs of the stakeholders using eHealth. CONCLUSION: Vocational and CPD training in General Practice needs to build on eHealth capabilities now. We strongly advise the incorporation of eHealth education into vocational training and CPD activities, rather than providing it as a separate single module. How learning goals and activities take shape and how competencies are evaluated clearly requires further practice, evaluation and study.

Perspectives of Patients and Professionals on Information and Education After Myocardial Infarction With Insight for Mixed Reality Implementation: Cross-Sectional Interview Study.

BACKGROUND: Patient education is crucial in the secondary prevention of cardiovascular disease. Novel technologies such as augmented reality or mixed reality expand the possibilities for providing visual support in this process. Mixed reality creates interactive digital three-dimensional (3D) projections overlaying virtual objects on the real-world environment. While augmented reality only overlays objects, mixed reality not just overlays but anchors virtual objects to the real world. However, research on this technology in the patient domain is scarce. OBJECTIVE: The aim of this study was to understand how patients perceive information provided after myocardial infarction and examine if mixed reality can be supportive in this process. METHODS: In total, 12 patients that experienced myocardial infarction and 6 health care professionals were enrolled in the study. Clinical, demographic, and qualitative data were obtained through semistructured interviews, with a main focus on patient experiences within the hospital and the knowledge they gained about their disease. These data were then used to map a susceptible timeframe to identify how mixed reality can contribute to patient information and education. RESULTS: Knowledge transfer after myocardial infarction was perceived by patients as too extensive, not personal, and inconsistent. Notably, knowledge on anatomy and medication was minimal and was not recognized as crucial by patients, whereas professionals stated the opposite. Patient journey analysis indicated the following four critical phases of knowledge transfer: at hospital discharge, at the first outpatient visit, during rehabilitation, and during all follow-up outpatient visits. Important patient goals were understanding the event in relation to daily life and its implications on resuming daily life. During follow-up, understanding physical limitations and coping with the condition and medication side effects in daily life emerged as the most important patient goals. The professionals' goals were to improve recovery, enhance medication adherence, and offer coping support. CONCLUSIONS: There is a remarkable difference between patients' and professionals' goals regarding information and education after myocardial infarction. Mixed reality may be a practical tool to unite perspectives of patients and professionals on the disease in a more even manner, and thus optimize knowledge transfer after myocardial infarction. Improving medication knowledge seems to be a feasible target for mixed reality. However, further research is needed to create durable methods for education on medication through mixed reality interventions.

Anatomy Dissection Course Improves the Initially Lower Levels of Visual-Spatial Abilities of Medical Undergraduates.

Visual-spatial abilities are considered a successful predictor in anatomy learning. Previous research suggest that visual-spatial abilities can be trained, and the magnitude of improvement can be affected by initial levels of spatial skills. This case-control study aimed to evaluate (1) the impact of an extra-curricular anatomy dissection course on visual-spatial abilities of medical undergraduates and (2) the magnitude of improvement in students with initially lower levels of visual-spatial abilities, and (3) whether the choice for the course was related to visual-spatial abilities. Course participants (n = 45) and controls (n = 65) were first and second-year medical undergraduates who performed a Mental Rotations Test (MRT) before and 10 weeks after the course. At baseline, there was no significant difference in MRT scores between course participants and controls. At the end of the course, participants achieved a greater improvement than controls (first-year: ∆6.0 ± 4.1 vs. ∆4.9 ± 3.2; ANCOVA, P = 0.019, Cohen's d = 0.41; second-year: ∆6.5 ± 3.3 vs. ∆6.1 ± 4.0; P = 0.03, Cohen's d = 0.11). Individuals with initially lower scores on the MRT pretest showed the largest improvement (∆8.4 ± 2.3 vs. ∆6.8 ± 2.8; P = 0.011, Cohen's d = 0.61). In summary, (1) an anatomy dissection course improved visual-spatial abilities of medical undergraduates; (2) a substantial improvement was observed in individuals with initially lower scores on the visual-spatial abilities test indicating a different trajectory of improvement; (3) students' preferences for attending extracurricular anatomy dissection course was not driven by visual-spatial abilities.

The Effect of Stereoscopic Augmented Reality Visualization on Learning Anatomy and the Modifying Effect of Visual-Spatial Abilities: A Double-Center Randomized Controlled Trial.

Monoscopically projected three-dimensional (3D) visualization technology may have significant disadvantages for students with lower visual-spatial abilities despite its overall effectiveness in teaching anatomy. Previous research suggests that stereopsis may facilitate a better comprehension of anatomical knowledge. This study evaluated the educational effectiveness of stereoscopic augmented reality (AR) visualization and the modifying effect of visual-spatial abilities on learning. In a double-center randomized controlled trial, first- and second-year (bio)medical undergraduates studied lower limb anatomy with stereoscopic 3D AR model (n = 20), monoscopic 3D desktop model (n = 20), or two-dimensional (2D) anatomical atlas (n = 18). Visual-spatial abilities were tested with Mental Rotation Test (MRT), Paper Folding Test (PFT), and Mechanical Reasoning (MR) Test. Anatomical knowledge was assessed by the validated 30-item paper posttest. The overall posttest scores in the stereoscopic 3D AR group (47.8%) were similar to those in the monoscopic 3D desktop group (38.5%; P = 0.240) and the 2D anatomical atlas group (50.9%; P = 1.00). When stratified by visual-spatial abilities test scores, students with lower MRT scores achieved higher posttest scores in the stereoscopic 3D AR group (49.2%) as compared to the monoscopic 3D desktop group (33.4%; P = 0.015) and similar to the scores in the 2D group (46.4%; P = 0.99). Participants with higher MRT scores performed equally well in all conditions. It is instrumental to consider an aptitude-treatment interaction caused by visual-spatial abilities when designing research into 3D learning. Further research is needed to identify contributing features and the most effective way of introducing this technology into current educational programs.

Endothelial dysfunction in pulmonary arterial hypertension: loss of cilia length regulation upon cytokine stimulation.

Pulmonary arterial hypertension (PAH) is a syndrome characterized by progressive lung vascular remodelling, endothelial cell (EC) dysfunction, and excessive inflammation. The primary cilium is a sensory antenna that integrates signalling and fine tunes EC responses to various stimuli. Yet, cilia function in the context of deregulated immunity in PAH remains obscure. We hypothesized that cilia function is impaired in ECs from patients with PAH due to their inflammatory status and tested whether cilia length changes in response to cytokines. Primary human pulmonary and mouse embryonic EC were exposed to pro- (TNFα, IL1ß, and IFNγ) and/or anti-inflammatory (IL-10) cytokines and cilia length was quantified. Chronic treatment with all tested inflammatory cytokines led to a significant elongation of cilia in both control human and mouse EC (by ∼1 µm, P < 0.001). This structural response was PKA/PKC dependent. Intriguingly, withdrawal of the inflammatory stimulus did not reduce cilia length. IL-10, on the other hand, blocked and reversed the pro-inflammatory cytokine-induced cilia elongation in healthy ECs, but did not influence basal length. Conversely, primary cilia of ECs from PAH patients were significantly longer under basal conditions compared to controls (1.86 ± 0.02 vs. 2.43 ± 0.08 µm, P = 0.002). These cilia did not elongate further upon pro-inflammatory stimulation and anti-inflammatory treatment did not impact cilia length. The missing length modulation was specific to cytokine stimulation, as application of fluid shear stress led to increased cilia length in the PAH endothelium. We identified loss of cilia length regulation upon cytokine stimulation as part of the endothelial dysfunction in PAH.

Fluid shear stress-induced TGF-ß/ALK5 signaling in renal epithelial cells is modulated by MEK1/2.

Renal tubular epithelial cells are exposed to mechanical forces due to fluid flow shear stress within the lumen of the nephron. These cells respond by activation of mechano-sensors located at the plasma membrane or the primary cilium, having crucial roles in maintenance of cellular homeostasis and signaling. In this paper, we applied fluid shear stress to study TGF-ß signaling in renal epithelial cells with and without expression of the Pkd1-gene, encoding a mechano-sensor mutated in polycystic kidney disease. TGF-ß signaling modulates cell proliferation, differentiation, apoptosis, and fibrotic deposition, cellular programs that are altered in renal cystic epithelia. SMAD2/3-mediated signaling was activated by fluid flow, both in wild-type and Pkd1 -/- cells. This was characterized by phosphorylation and nuclear accumulation of p-SMAD2/3, as well as altered expression of downstream target genes and epithelial-to-mesenchymal transition markers. This response was still present after cilia ablation. An inhibitor of upstream type-I-receptors, ALK4/ALK5/ALK7, as well as TGF-ß-neutralizing antibodies effectively blocked SMAD2/3 activity. In contrast, an activin-ligand trap was ineffective, indicating that increased autocrine TGF-ß signaling is involved. To study potential involvement of MAPK/ERK signaling, cells were treated with a MEK1/2 inhibitor. Surprisingly, fluid flow-induced expression of most SMAD2/3 targets was further enhanced upon MEK inhibition. We conclude that fluid shear stress induces autocrine TGF-ß/ALK5-induced target gene expression in renal epithelial cells, which is partially restrained by MEK1/2-mediated signaling.

Measuring the primary cilium length: improved method for unbiased high-throughput analysis.

BACKGROUND: Primary cilia are cellular protrusions involved in mechanic and chemical sensing on almost all cells of our body. Important signaling pathways, including Hedgehog, TGFß, and Ca(2+), are linked to cilia and/or cilia function. Cilia can vary in length, which has functional implications. To measure these lengths correctly, a standardized method with high reliability and throughput is required. To date, methods for length measurements in cultured cells after fluorescent staining for ciliary components are error prone with a possible human selection bias, primarily caused by the orientation of cilia with respect of the imaging plane. In tissue sections, accurate measurements become an even larger challenge due to additional random sectioning plane. Cilia can be reconstructed in 3D and measured one by one, but this is a labor-intensive procedure. Therefore, we developed a new, high-throughput method with less selection bias. RESULTS: To identify the optimal type of measurement of straight and relatively short cilia, three methods were compared. The first method is based on maximum intensity projection (MIP), the second method is based on the Pythagorean theorem (PyT), and the third is based on 3D alternative angled slicing (DAAS). We investigated whether cilia visible in the plane of focus ('flat cilia'), and the ones that are angled with respect to the plane of focus are represented differently among the various methods. To test the agreement between the methods, intraclass correlations are calculated. To measure flat cilia, MIP and DAAS provided representative results, with the MIP method allowing for higher throughput. However, when measuring the angled cilia with MIP, the actual cilium length is overtly underestimated. DAAS and PyT are exchangeable methods for length measurements of the angled cilia, while PyT exhibits higher throughput and is therefore the preferred method for measuring the length of an angled cilium. CONCLUSION: PyT is a universal measuring method to measure straight cilia, without selection bias. MIP provides similar results for flat cilia, but underestimates the length of angled cilia. In addition, PyT facilitates high-throughput length measurements. Manual tracking or reconstruction will be the method of choice to measure irregularly shaped cilia.

Common arterial trunk and ventricular non-compaction in Lrp2 knockout mice indicate a crucial role of LRP2 in cardiac development.

Lipoprotein-related receptor protein 2 (LRP2) is important for development of the embryonic neural crest and brain in both mice and humans. Although a role in cardiovascular development can be expected, the hearts ofLrp2knockout (KO) mice have not yet been investigated. We studied the cardiovascular development ofLrp2KO mice between embryonic day 10.5 (E10.5) and E15.5, applying morphometry and immunohistochemistry, using antibodies against Tfap2α (neural crest cells), Nkx2.5 (second heart field), WT1 (epicardium derived cells), tropomyosin (myocardium) and LRP2. TheLrp2KO mice display a range of severe cardiovascular abnormalities, including aortic arch anomalies, common arterial trunk (persistent truncus arteriosus) with coronary artery anomalies, ventricular septal defects, overriding of the tricuspid valve and marked thinning of the ventricular myocardium. Both the neural crest cells and second heart field, which are essential for the lengthening and growth of the right ventricular outflow tract, are abnormally positioned in theLrp2KO. This explains the absence of the aorto-pulmonary septum, which leads to common arterial trunk and ventricular septal defects. Severe blebbing of the epicardial cells covering the ventricles is seen. Epithelial-mesenchymal transition does occur; however, there are fewer WT1-positive epicardium-derived cells in the ventricular wall as compared to normal, coinciding with the myocardial thinning and deep intertrabecular spaces. LRP2 plays a crucial role in cardiovascular development in mice. This corroborates findings of cardiac anomalies in humans withLRP2mutations. Future studies should reveal the underlying signaling mechanisms in which LRP2 is involved during cardiogenesis.

Environmental Influences on Endothelial to Mesenchymal Transition in Developing Implanted Cardiovascular Tissue-Engineered Grafts.

Tissue-engineered grafts for cardiovascular structures experience biochemical stimuli and mechanical forces that influence tissue development after implantation such as the immunological response, oxidative stress, hemodynamic shear stress, and mechanical strain. Endothelial cells are a cell source of major interest in vascular tissue engineering because of their ability to form a luminal antithrombotic monolayer. In addition, through their ability to undergo endothelial to mesenchymal transition (EndMT), endothelial cells may yield a cell type capable of increased production and remodeling of the extracellular matrix (ECM). ECM is of major importance to the mechanical function of all cardiovascular structures. Tissue engineering approaches may employ EndMT to recapitulate, in part, the embryonic development of cardiovascular structures. Improved understanding of how the environment of an implanted graft could influence EndMT in endothelial cells may lead to novel tissue engineering strategies. This review presents an overview of biochemical and mechanical stimuli capable of influencing EndMT, discusses the influence of these stimuli as found in the direct environment of cardiovascular grafts, and discusses approaches to employ EndMT in tissue-engineered constructs.

SLUG is expressed in endothelial cells lacking primary cilia to promote cellular calcification.

OBJECTIVE: Arterial calcification is considered a major cause of death and disabilities worldwide because the associated vascular remodeling leads to myocardial infarction, stroke, aneurysm, and pulmonary embolism. This process occurs via poorly understood mechanisms involving a variety of cell types, intracellular mediators, and extracellular cues within the vascular wall. An inverse correlation between endothelial primary cilia and vascular calcified areas has been described although the signaling mechanisms involved remain unknown. We aim to investigate the signaling pathways regulated by the primary cilium that modulate the contribution of endothelial cells to vascular calcification. APPROACH AND RESULTS: We found that human and murine endothelial cells lacking primary cilia are prone to undergo mineralization in response to bone morphogenetic proteins stimulation in vitro. Using the Tg737(orpk/orpk) cillium-defective mouse model, we show that nonciliated aortic endothelial cells acquire the ability to transdifferentiate into mineralizing osteogenic cells, in a bone morphogenetic protein-dependent manner. We identify ß-CATENIN-induced SLUG as a key transcription factor controlling this process. Moreover, we show that the endothelial expression of SLUG is restricted to atheroprone areas in the aorta of LDLR(-/-) mice. Finally, we demonstrate that SLUG and phospho-homolog of the Drosophila protein, mothers against decapentaplegic (MAD) and the Caenorhabditis elegans protein SMA (from gene sma for small body size)-1/5/8 expression increases in endothelial cells constituting the vasa vasorum in the human aorta during the progression toward atherosclerosis. CONCLUSIONS: We demonstrated that the lack of primary cilia sensitizes the endothelium to undergo bone morphogenetic protein-dependent-osteogenic differentiation. These data emphasize the role of the endothelial cells on the vascular calcification and uncovers SLUG as a key target in atherosclerosis.

Accurate blood flow measurements: are artificial tracers necessary?

Imaging-based blood flow measurement techniques, such as particle image velocimetry, have become an important tool in cardiovascular research. They provide quantitative information about blood flow, which benefits applications ranging from developmental biology to tumor perfusion studies. Studies using these methods can be classified based on whether they use artificial tracers or red blood cells to visualize the fluid motion. We here present the first direct comparison in vivo of both methods. For high magnification cases, the experiments using red blood cells strongly underestimate the flow (up to 50% in the present case), as compared to the tracer results. For medium magnification cases, the results from both methods are indistinguishable as they give the same underestimation of the real velocities (approximately 33%, based on in vitro reference measurements). These results suggest that flow characteristics reported in literature cannot be compared without a careful evaluation of the imaging characteristics. A method to predict the expected flow averaging behavior for a particular facility is presented.

Shear induced collateral artery growth modulated by endoglin but not by ALK1.

Transforming growth factor-beta (TGF-ß) stimulates both ischaemia induced angiogenesis and shear stress induced arteriogenesis by signalling through different receptors. How these receptors are involved in both these processes of blood flow recovery is not entirely clear. In this study the role of TGF-ß receptors 1 and endoglin is assessed in neovascularization in mice. Unilateral femoral artery ligation was performed in mice heterozygous for either endoglin or ALK1 and in littermate controls. Compared with littermate controls, blood flow recovery, monitored by laser Doppler perfusion imaging, was significantly hampered by maximal 40% in endoglin heterozygous mice and by maximal 49% in ALK1 heterozygous mice. Collateral artery size was significantly reduced in endoglin heterozygous mice compared with controls but not in ALK1 heterozygous mice. Capillary density in ischaemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared with controls. To provide mechanistic evidence for the differential role of endoglin and ALK1 in shear induced or ischaemia induced neovascularization, murine endothelial cells were exposed to shear stress in vitro. This induced increased levels of endoglin mRNA but not ALK1. In this study it is demonstrated that both endoglin and ALK1 facilitate blood flow recovery. Importantly, endoglin contributes to both shear induced collateral artery growth and to ischaemia induced angiogenesis, whereas ALK1 is only involved in ischaemia induced angiogenesis.

TGF-ß signaling in endothelial-to-mesenchymal transition: the role of shear stress and primary cilia.

Endothelial-to-mesenchymal transition (EndoMT) is an instrumental step in the development of valves in the embryonic heart. This process is driven by activation of transforming growth factor-ß (TGF-ß) signaling and is characterized by the loss of endothelial and gain of mesenchymal phenotype, and by delamination of cells from the surface into the underlying endocardial cushion matrix. The endothelial cells (ECs) overlying the cushions are typically exposed to high blood flow and concomitant shear stress and do not have a primary cilium. Here, we show that shear stress activates TGF-ß-Alk5 signaling in ECs, which is necessary for EndoMT in the cushions. Moreover, we show that the absence of a primary cilium is critically important for this transition process.