Knockdown of TPI in human dermal microvascular endothelial cells and its impact on angiogenesis in vitro.

INTRODUCTION: Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs. OBJECTIVE: The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined. METHODS: Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored. RESULTS: Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis. CONCLUSION: Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.

Outcome of Communication Training in Veterinary Studies: Influence on the Perception of the Relevance of Veterinary Competencies and Self-Assessment of Communication Skills.

Since communication skills contribute significantly to professional success among veterinarians, there is a particular focus on developing communication classes in veterinary curricula. At Freie Universität Berlin, an e-learning course covering the basics of communication and a practical communication course based on role plays with and without simulation persons have been established. The outcome of these communication courses on the assessment of the relevance of several veterinary competencies and on the self-assessment of communication skills using the SE-12 questionnaire was investigated. For this purpose, students were surveyed before and after the e-learning course as well as before and after the practical course. Veterinarians were also surveyed on the relevance of veterinary competencies. The relevance of communicative competencies for professional success was rated significantly higher by the students after completing the practical course than by the other students and the veterinarians. Self-assessment of communication skills showed little increase after the e-learning course, but a significant increase after the practical course. Thus, an effective outcome of the communication classes was observed mainly after the practical course. However, the effect of the e-learning course cannot be ruled out since the students participating in the practical course have also completed the e-learning course beforehand.

Depression, suicidal ideation and suicide risk in German veterinary medical students compared to the German general population.

BACKGROUND: Various studies from different countries indicated that veterinarians have a significantly increased risk of depression, suicidal ideation and of death by suicide. For German veterinarians a recent study has demonstrated a three times higher rate for depression, two times higher rate for suicidal ideation and a five times higher suicide risk compared to the German general population. For veterinary students, recent studies in the US and UK indicated higher mental distress. To date, empirical studies on depression, suicidal ideation and suicide risk among veterinary students in Germany were lacking so far. This study investigates depression, suicidal ideation and suicide risk of veterinary students in Germany. METHODS: 913 German veterinary students (14.3% response rate, 90.7% women, mean age 23.6 years) between 18 and 46 years were included and compared with representative German general population samples from 2007 (N = 1097, 55.4% women, mean age 33.9) and 2015 (N = 1033, 56.1% women, mean age 32.8) of the same age range using the depression module of the Patient Health Questionnaire (PHQ-9) and Suicide Behaviors questionnaire-Revised (SBQ-R). The general population samples were collected with the assistance of a demographic consulting company. RESULTS: The prevalence of depression among German veterinary students was 45.9% (compared to 3.2% in the general population), suicidal ideation was 19.9% (compared to 4.5% in the general population) and suicide risk was 24.0% (compared to 6.6% in the general population). CONCLUSION: In this study, German veterinary students have a 22.1 times higher risk to be screened positive for depression, a 4 times higher risk for reporting current suicidal ideation and they are 4.2 times more likely to have an increased suicide risk compared with the general population in Germany of the same age range.

Expression of vimentin, TPI and MAT2A in human dermal microvascular endothelial cells during angiogenesis in vitro.

INTRODUCTION: In vitro assays of angiogenesis face immense problems considering their reproducibility based on the inhomogeneous characters of endothelial cells (ECs). It is necessary to detect influencing factors, which affect the angiogenic potency of ECs. OBJECTIVE: This study aimed to analyse expression profiles of vimentin (VIM), triosephosphate isomerase (TPI) and adenosylmethionine synthetase isoform type-2 (MAT2A) during the whole angiogenic cascade in vitro. Furthermore, the impact of knocking down vimentin (VIM) on angiogenesis in vitro was evaluated, while monitoring TPI and MAT2A expression. METHODS: A long-term cultivation and angiogenic stimulation of human dermal microvascular ECs was performed. Cells were characterized via VEGFR-1 and VEGFR-2 expression and a shRNA-mediated knockdown of VIM was performed. The process of angiogenesis in vitro was quantified via morphological staging and mRNA-and protein-levels of all proteins were analysed. RESULTS: While native cells ran through the angiogenic cascade chronologically, knockdown cells only entered beginning stages of angiogenesis and died eventually. Cell cultures showing a higher VEGFR-1 expression survived exclusively and displayed an upregulation of MAT2A and TPI expression. Native cells highly expressed VIM in early stages, MAT2A mainly in the beginning and TPI during the course of angiogenesis in vitro. CONCLUSION: VIM knockdown led to a deceleration of angiogenesis in vitro and knockdown cells displayed expressional changes in TPI and MAT2A. Cell populations with a higher number of stalk cells emerged as being more stable against manipulations and native expression profiles provided an indication of VIM and MAT2A being relevant predominantly in beginning stages and TPI during the whole angiogenic cascade in vitro.

Teaching Outbreak Investigations with an Interactive Blended Learning Approach.

Public health is a central but often neglected component of veterinary education. German veterinary public health (VPH) education includes substantial theory-focused lectures, but practical case studies are often missing. To change this, we combined the advantages of case-based teaching and blended learning to teach these topics in a more practical and interactive way. Blended learning describes the combination of online and classroom-based teaching. With it, we created an interdisciplinary module for outbreak investigations and zoonoses, based on the epidemiology, food safety, and microbiology disciplines. We implemented this module within the veterinary curriculum of the seventh semester (in the clinical phase of the studies). In this study, we investigated the acceptance of this interdisciplinary approach and established a framework for the creation of interactive outbreak investigation cases that can serve as a basis for further cases. Over a period of 3 years, we created three interactive online cases and one interactive in-class case and observed the student-reported evaluation of the blended learning concept and self-assessed learning outcomes. Results show that 80% (75-89) of students evaluated the chosen combination of case-based and blended learning for interdisciplinary teaching positively and therefore accepted it well. Additionally, 76% (70-98) of students evaluated their self-assessed learning outcomes positively. Our results suggest that teaching VPH through interdisciplinary cases in a blended learning approach can increase the quality of teaching VPH topics. Moreover, it provides a framework to incorporate realistic interdisciplinary VPH cases into the curriculum.

Task force veterinary anatomy: joint efforts of the five German veterinary schools to ensure education during the COVID-19 pandemic.

At the start of the COVID-pandemic in March 2020, the Institutes of Veterinary Anatomy of the five German educational institutions were confronted with the challenge of digitalising all lectures for the second and fourth semesters of veterinary students. After an online kick-off event and a preliminary status quo meeting, available digital teaching material was exchanged for students to stream from learning platforms. Lectures were either synchronized or made available as audio recordings and connotated slides on the learning platforms. Fortunately, digital microscopic slides had already been in use, which made it easy for students to access them. Dissection exercises mostly consisted of self-study, using instructive videos and interactive exercises. In the second half of the semester, four of the educational institutions were able to offer a restricted number of in-person gross anatomy classes under reinforced conditions. Success monitoring took place online through different formats, and partially on a voluntary basis, via the learning platforms. Although the past two semesters had to almost exclusively take place online due to the unprecedented circumstances, and joint efforts of the five veterinary institutions, there is a general consensus that the practical education in anatomy, histology and embryology is essential to veterinary students. In fact, it is the only way they can obtain the necessary skills to successfully complete the rest of their degree.

Communication as teaching content of veterinary studies - a joint position paper from the DVG specialist group "communication and didactics" and the GMA veterinary medicine committee.

Veterinarians have to face many communicative challenges in their professional capacity. Successful professional communication increases satisfaction among pet owners, staff members and colleagues, and ultimately also the veterinarians' own satisfaction. On the other hand, unsuccessful communication can easily lead to rejection, distrust and dissatisfaction. However, communicative skills are not explicitly taught as part of the compulsory courses in veterinary medicine in Germany. The position paper of the GMA Veterinary Medicine Committee and the DVG work group Didactics and Communication Competence describes the importance of successful communication for working veterinarians as well as the training situation in Germany and addresses topics that are often discussed in connection with the implementation. The authors come to the conclusion that there is both a necessity and a possibility for the introduction of communicative training content and provide recommendations that are intended to support the sustainable introduction of courses and exams to develop the communicative skills of veterinary students.

Integration and potential of teaching communication skills in the study of veterinary medicine in Germany.

Goal: Presentation of the current range of courses regarding communication at the five German educational institutions for veterinary medicine. In addition to learning objectives and individual solutions, possible potential for future developments are presented. Methods: Interviews with communication educators at the five German education institutions and subsequent synopsis. Results: To date, there are no binding education guidelines regarding communication in veterinary medicine. Nevertheless, communication education has been introduced at all five education institutions, albeit depth and formats vary considerably. The learning objectives are largely consistent and based on the recommendations for day-one-skills made by the European Association of Establishments for Veterinary Education. Communication is not recognized as a fully-fledged subject in the curricula of any of the education institutions. All education institutions clearly fall short of teaching the recommended 150 lecture hours. Conclusion: To ensure communication skills in veterinary medicine graduates, binding education guidelines should be agreed upon. Communication education should be integrated into all veterinary curricula as a fully-fledged subject with longitudinally increasing depth.

Euthanasia Distress and Fearlessness About Death in German Veterinarians.

Background: The elevated risk of suicide in veterinarians is still not well understood, but euthanasia distress and increased fearlessness about death (FAD) have been discussed as specific risk factors. Aims: We investigated German veterinarians to test whether FAD is increased compared with the general population, whether FAD is associated with euthanasia distress, and whether there are differences between different fields in veterinary medicine. Methods: German veterinarians were assessed via self-report instruments and compared with a representative general population sample. There was no difference in FAD. Lower euthanasia distress was significantly associated with higher FAD. Limitations: Although we were able to recruit a large sample of veterinarians, the generalizability is limited by the voluntary nature of participation and the related risk of bias. Results: There was no difference in FAD. Lower euthanasia distress was significantly associated with higher FAD. Conclusion: Although lower euthanasia distress as an indicator of habituation to euthanasia is associated with higher FAD, one cannot assume that increased FAD in veterinarians is a specific risk factor for suicidality in veterinarians.

Depression, suicidal ideation and suicide risk in German veterinarians compared with the general German population.

BACKGROUND: Higher rates of depression, suicidal ideation and suicide risk have been reported for veterinarians in various studies worldwide. This study investigates whether this is also true for German veterinarians. METHODS: A total of 3.118 veterinarians (78.8 per cent female, mean age 41.3 years) between 22 and 69 years were included and compared with two general population samples of the same age range using the Suicide Behaviours Questionnaire-Revised and Patient Health Questionnaire. RESULTS: Current suicidal ideation was found in 19.2 per cent of veterinarians, compared with only 5.7 per cent in the general population. 32.11 per cent of veterinarians were classified with increased suicide risk, compared with 6.62 per cent in the general population. 27.78 per cent of veterinarians screened positive for depression, compared with 3.99 per cent of the general population. CONCLUSION: The study shows that veterinarians have an increased risk of depression and suicidal ideation and suicide risk compared with the general population in Germany. Similar to previous findings, the level of depression was higher among veterinarians than in the general population. However, this study does not explore causes for higher rates in depression, suicide risk and suicidal ideation. Since other studies strongly suggest specific risk factors lead to higher suicide risk and consequently elevated numbers of completed suicides, future research should focus on identifying and preventing causes.

Quantitation of Tumor Angiogenesis In Vitro: An All-In-One Angiogenesis Assay.

In vitro angiogenesis systems enable the analysis of pro- or anti-angiogenic compounds. Most in vitro models do not reproduce the entire angiogenic cascade, from cell migration and proliferation to tube formation. Here, we describe an all-in-one angiogenesis assay that mimics the entire angiogenic cascade in vitro, rendering this model an ideal tool for the in vitro testing.

Test Anxiety: Evaluation of a Low-Threshold Seminar-Based Intervention for Veterinary Students.

Veterinary students are confronted with a high workload and an extensive number of examinations. However, the skills students gained in high school cannot serve as satisfactory coping strategies during veterinary training. This disparity can lead to test anxiety, as frequently reported by international surveys. In response, a pilot study was carried out to evaluate the effects of a newly developed training seminar to prevent and/or reduce test anxiety. The seminar was offered on a voluntary basis as a low-threshold intervention to first- and second-year veterinary students at three different veterinary schools in Germany. The intervention was offered in two different designs: in either a block or in a semester course containing cognitive and behavioral approaches as well as skill-deficit methods. By conducting a survey and interviews among the participants it was determined whether the contents of the seminar were perceived as helpful for counteracting test anxiety. The potential of the intervention was evaluated using a German test anxiety questionnaire (PAF). The contents of the training seminar were all assessed as beneficial but evaluated slightly differently by first- and second-year students. The results indicate that the seminar prevents and reduces test anxiety significantly compared to the control group students. The greatest effects were achieved by offering the intervention to first-year students and as a block course. As the participants benefit from the intervention independent of the extent of test anxiety, these results suggest that it may be profitable to integrate a workshop on coping strategies in the veterinary curriculum.

Searching for markers to identify angiogenic endothelial cells: a proteomic approach.

In the field of angiogenesis research considerable effort is put in the development of in vitro assays of angiogenesis to replace animal experiments. Unfortunately, reproducibility of these assays frequently fails depending on the particular batch of endothelial cells delivered by the distributor. This is due to the lack of reliable markers for the identification and isolation of angiogenic microvascular endothelial cells that have the capacity to perform all stages of the angiogenic cascade. This study was carried out to identify potential markers for angiogenic versus non-angiogenic endothelial cells. The protein expression profile of four capillary-derived human microvascular primary endothelial cell cultures of which only two batches could be stimulated to angiogenesis was investigated and compared by two-dimensional gel electrophoresis. Seven proteins were found to be expressed in the angiogenic batches only. One protein was detected exclusively in the non-angiogenic batches. These proteins might be verified as markers for angiogenic endothelial cells.

Intussusceptive angiogenesis: a biologically relevant form of angiogenesis.

Angiogenesis, i.e. the development and growth of blood vessels, is a major topic of research as it plays an important role in normal development and in various pathologies. Recent evidence revealed the existence of different mechanisms of blood vessel growth, including sprouting and intussusceptive angiogenesis, vascular mimicry, and blood vessel cooption. The latter two have only been observed in tumor growth, but sprouting and intussusceptive angiogenesis also occur in healthy, physiologically growing tissues. Despite this variety of angiogenic mechanisms, most of the current research is focused on the mechanism of sprouting angiogenesis because this mechanism was first described and because most existing experimental models are related to sprouting angiogenesis. Consequently, the mechanism of intussusceptive angiogenesis is often overlooked in angiogenesis research. Here, the mechanism of intussusceptive angiogenesis is reviewed and the current techniques and models for investigating intussusceptive angiogenesis are summarized. In addition, other mechanisms of vascular growth are briefly reviewed.

Angiopoietins differentially influence in vitro angiogenesis by endothelial cells of different origin.

Angiopoietins are important growth factors for vascular development and quiescence. They are promising targets for pro- or anti-angiogenic therapies in diverse pathologies, but the mechanisms of the ANGPT/TIE2 system are complex and not well understood. In the present study, the separate and combined effects of angiopoietin 1 and angiopoietin 2 were studied, using a recently developed in vitro angiogenesis model that allows both a quantitative and qualitative evaluation of the angiogenic cascade. This cell culture model was performed with microvascular endothelial cells (ECs) originating from different vascular beds, i.e. dermal ECs and cardiac ECs. In addition, the expression of the angiopoietins and the receptors, TIE1 and TIE2 was analyzed with RT-qPCR. This study revealed that the angiopoietins provoked a differential response in the two endothelial cultures. Both angiopoietin 1 as well as angiopoietin 2 elicited an angiogenic cascade in the dermal ECs but not in the cardiac ECs. In addition, the RT-qPCR data revealed marked differences in the endogenous expression pattern of these factors, indicating that the origin of endothelial cells might have an important impact on their angiogenic potential.

In vitro angiogenic potency in human microvascular endothelial cells derived from myocardium, lung and skin.

Human microvascular endothelial cells derived from myocardium (HCMEC), lung (HPMEC) and foreskin (HDMEC) showed different angiogenic potency when cultivated in their original growth media provided by the distributors. In order to standardize microenvironmental conditions in an all-in-one assay of angiogenesis the aim of this study was to find one optimal growth medium for the endothelial cells derived from the different organs. Therefore each endothelial cell type was cultivated under identical conditions in the different original growth media as well as in several media formulations of the original growth media. Results reveal that even if cultivated in the same growth medium under exactly the same cultivation conditions--over a prolonged time period of 60 days--the endothelial cells still showed different angiogenic potency. This is due to a combination of extrinsic factors, i.e. the isolation procedure and in particular the growth medium, as well as to intrinsic differences between cells of diverse origin.

Quantitation of angiogenesis in vitro induced by VEGF-A and FGF-2 in two different human endothelial cultures - an all-in-one assay.

Angiogenic therapy is considered to be a promising tool for treatment of ischemic diseases. Many in vivo and in vitro assays have been developed to identify potential proangiogenic drugs and to investigate their mode of action. However, until now no validated system exists that would allow quantitation of angiogenesis in vitro in only one assay. Here, a previously established all-in-one in vitro assay based on staging of the angiogenic cascade was validated by quantitation of the effects of the known proangiogenic factors VEGF-A and FGF-2. Both growth factors were applied separately or in combination to human endothelial cell cultures derived from the heart and the foreskin, and angiogenesis was quantitated over 30 days of culture. Additionally, gene expression of VEGFR-1, VEGFR-2 and FGFR-1 at 3, 10, 20 or 40 days of cultivation was quantitated by RT-qPCR. In both cultures, VEGF-A as well as FGF-2 induced a run through all defined stages of angiogenesis in vitro. Application of VEGF-A only led to formation of irregular globular endothelial structures, while FGF-2 resulted in development of regular capillary-like structures. Quantitation of the angiogenic effects of VEGF-A and transcripts of VEGFR-1 and VEGFR-2 showed that a high VEGFR-1/VEGFR-2 ratio evoked deceleration of angiogenesis.

The glycocalyx of human, bovine and murine microvascular endothelial cells cultured in vitro.

This study investigated the morphology and thickness of the glycocalyx linings of microvascular endothelial cells (MVEC). Three distinct cell types were used: the human dermal cells (HDMVEC), the murine cardiac cells (MCMVEC) and the bovine luteal cells (BLMVEC). Cells were cultivated for 48 h. Glycocalyx was stained with ruthenium red and examined under a transmission electron microscope. The glycocalyx of HDMVEC was thin and constant (10-22 nm). No glycocalyx was detected within intracellular vesicles. Two cell populations of MCMVEC were recorded. The minor MCMVEC population was well differentiated and covered with heterogenous glycocalyx (2-200 nm). Conglomerates formed above the baseline along the cell extensions. The major MCMVEC population was undifferentiated and coated by a smooth and thin (12-25 nm) layer of glycocalyx. Intracellular vesicles were also coated with glycocalyx. In the BLMVEC population, 10% had 3-170 nm of discontinuous glycocalyx. Rough conglomerates were observed along cell sprouts. Their intracellular vesicles were coated with glycocalyx. The study found vast differences in the morphology and thickness of endothelial glycocalyx among different MVEC under in vitro cultivation. The only record of active endocytosis was in BLMVEC and MCMVEC. No evidence of active endocytosis was found in HDMVEC.

Quantitation of angiogenesis and antiangiogenesis in vivo, ex vivo and in vitro - an overview.

Angiogenesis is defined as the sprouting of new capillaries from pre-existing ones. Angiogenesis is a prerequisite for growth and differentiation of organs and tissues and is involved in many pathological processes, for example growth and metastasis of tumours. Numerous in vivo and in vitro models of angiogenesis have been developed in order to identify and analyse pro- and antiangiogenic factors. This review aims at outlining the topic of quantitation of angiogenesis and antiangiogenesis in these models.

Vascular dynamics in relation to immunolocalisation of VEGF-A, VEGFR-2 and Ang-2 in the bovine corpus luteum.

Vascular dynamics during development and regression of the bovine corpus luteum were investigated morphometrically in relation to immunolocalisation of vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor receptor 2 (VEGFR-2) and angiopoietin 2 (Ang-2) in the different cell types. Angiogenesis and remodelling of the capillary network between day 8 and 17 of the oestrous cycle was driven by luteal cells, which were highly immunopositive for VEGF-A. Thereafter, immunoreactivity for VEGF-A and VEGFR-2 was mainly found in vascular smooth muscle cells, the predominant cell type in late luteolysis. During early regression, apoptosis of luteal and endothelial cells was closely correlated, resulting in a significant decrease of capillarity. In late regression, an increase in capillary density was found, suggesting that regression and transformation into the corpus albicans requires adequate perfusion. In the phases of vascular remodelling, i.e. mature and late regressing corpus luteum, high scores of Ang-2-immunopositive endothelial and smooth muscle cells were found. Therefore, it may be hypothesised that Ang-2 supports the angiogenic effects of VEGF-A in these luteal stages. Results emphasise that precise staging and a differentiated view on the cellular populations is important in evaluating the controlled regression and transformation of the corpus luteum.