Growing blood vessels to treat limb ischemia : studie in mice and man

Summary This thesis describes the efforts of increasing our knowledge and insights into cellular and molecular mechanisms of vascular growth, especially collateral artery growth (arteriogenesis), in limb ischemia with the aim of developing new strategies for therapeutic angiogenesis and arteriogenesis. Conclusions are drawn from experiences in a mouse model mimicking peripheral artery disease, biopsies from human amputated limbs, and a trial using angiogenic gene therapy in patients. Chapter 1 reviews what is known to date about the cellular and molecular mechanisms of vascular growth, with particular emphasis on the role of angiogenic factors, the immune system, and bone marrow. Furthermore, results and limitations of previous therapeutic angiogenesis trials for treatment of peripheral artery disease are reviewed. In conclusion, disappointing results from recent placebo-controlled trials merit more basic research in this field, with focus on optimizing cell-based therapies. The large variety of surgical techniques and end point measurements that are applied between studies using ischemic hind limb models are discussed in Chapter 2. A guideline is provided on how to interpret results from these studies depending on the model used. In this thesis, a mouse model was selected to study the development of collateral arteries by selecting an appropriate surgical approach, using detailed angiography to visualize collaterals and determining their hemodynamic significance by perfusion measurement. In Chapter 3, the unanticipated finding is described that mouse strains with different bias in immune responsiveness show major differences in collateral forming capacity. Subsequently, it was hypothesized that lymphocytes play a role in collateral formation. Arteriogenesis was impaired in mice lacking natural killer (NK) cells, however not in mice lacking natural killer T (NKT)-cells (a regulatory subset of T-cells). Furthermore, arteriogenesis was impaired in mice lacking CD4+ T-cells. This impairment was even more profound if these mice were depleted of NK-cells. In addition, evidence was provided that T-cells and NK-cells accumulate around collateral arteries, and secrete a variety of inflammatory cytokines. In conclusion, these data show that NK-cells and CD4+ T-cells are involved in collateral formation in mice. Stimulation of arteriogenesis by specific activation of defined lymphocyte subsets might be a promising treatment for patients with ischemic disease. Chapter 4 provides evidence in human ischemic skeletal muscle for a role of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) in adult neovascularization via retention of CXCR4-positive cells. Moreover, VEGF, SDF-1 and CXCR4 expressions in ischemic muscle were up-regulated in 2 patients with acute-on-chronic ischemia, whereas down-regulated in 9 of 13 patients with chronic ischemia. This may be explained by an inability of hypoxic tissues to sufficiently express the transcription factor hypoxia inducible factor 1_ (HIF-1_) in chronic ischemia. These data are the first to show in humans a pivotal role of SDF-1 in the retention of bone marrow-derived cells in hypoxic tissues. Furthermore, future experiments aiming on differences in angiogenic expression profile between acute and chronic hypoxic conditions may lead to optimized angiogenic treatments for patients with chronic ischemic disease. In Chapter 5, it was shown that there was no impairment of angiographic collateral formation and only limited impairment of perfusion recovery in diabetic or insulin-resistant mice. Collateral formation was, however, severely impaired in hypercholesterolemic mice fed on high-fat diets. There was an inverse correlation of perfusion recovery with plasma cholesterol levels, but not with triglyceride, free fatty acid, glucose or insulin levels. In conclusion, impairment of arteriogenesis is more associated with hyperlipidemia than hyperglycemia or hyperinsulinemia, and is cholesterol-dependent in mice. In line with this, evidence is accumulating that a disturbed lipid metabolism is a crucial determinant of the development of diabetes and its complications. Therefore, a disturbed lipid profile might be crucial for the impairment of collateral formation in diabetic patients, stressing the importance of lipid-lowering drugs to prevent complications of diabetes. In Chapter 6, it was demonstrated that VEGF gene therapy results in enhanced expression of myoglobin, a protein that plays an important role in oxygen metabolism of muscle cells, in ischemic skeletal muscle in mice. Furthermore, we show co-expression of VEGF and myoglobin in muscle biopsies from patients after limb amputation caused by peripheral arterial disease, which correlates with the degree of ischemia. In addition, a direct regulation of myoglobin by VEGF was shown in murine myotubes in culture. Our data indicate that VEGF therapy, apart from inducing new capillaries, changes properties of skeletal muscle fibers resulting in improved muscle oxygenation, which may explain the puzzling inconsistencies shown in previous clinical trials with VEGF. VEGF-mediated increase of muscle myoglobin may clarify, at least partly, the observed clinical improvements of VEGF-treated patients in the absence of improved vascular status. Chapter 7 consists of the results from a double-blind randomized trial comparing intramuscular VEGF165 plasmid treatment (N=27) with placebo (N=27) in diabetic patients with critical limb ischemia. The primary end point was the amputation rate at 100 days. Secondary end points were a 15% increase in pressure indices between ankle and arm, clinical improvement and safety. In VEGF- versus placebo-treated patients, amputation occurred in 3 versus 6 (NS), hemodynamics improved in 7 versus 1 (P=0.05), skin ulcers healed in 7 versus 0 (P=0.01), and pain decreased in 5 versus 2 (NS). No substantial adverse effects were observed. Although this trial failed to meet the primary objective of significant amputation reduction, VEGF gene therapy resulted in some significant clinical improvements. Hopefully, this study could serve to regenerate interest in therapeutic angiogenesis after recent disappointing trials. Conclusions In this thesis a mouse model was used to study the cellular and molecular mechanisms of arteriogenesis. Only recently, one has begun to unravel the role of the immune system and its cellular components in collateral artery growth. Here, evidence is provided for a role of lymphocytes. More insights into which (other) cell types are involved will enlarge the toolbox for stimulation of arteriogenesis and may refine autologeous bone-marrow transplantation, as recently applied in clinics, for instance by administration of defined lymphocyte subsets or their specific activation/inhibition with ligands for activating or inhibitory receptors, respectively. Using the mouse model, knowledge may not only be brought from bench to bedside, but also from bedside to bench. For instance, the unexplained beneficial results in VEGF-treated patients without improved vascularization led to the hypothesis that VEGF may not only simply __grow vessels__, but may also improve muscle oxygenation by changing muscle composition, which was then proven in mice. This finding in turn holds considerable promise for the development of novel therapeutics to treat various diseases. Nevertheless, it is important to realize that mice are not patients. Although genetically modified mice mimic disease profiles of patients, for instance by inducing dyslipidemia or diabetes, vascular disease in patients seems more complex; multiple vascular risk factors coexist, and endothelium, bone marrow and/or the immune system may be dysfunctional, leading to impaired arteriogenesis. Moreover, in this thesis evidence is provided for an inability of hypoxic tissues to express angiogenic factors in patients with chronic ischemia. More insights into impaired vascular growth in patients are important, since most placebo-controlled trials with angiogenic or arteriogenic factors were negative to date. Various reasons, ranging from the type of angiogenic factor to technique of administration to patient selection may account for this. The design of the trial described in this thesis using intramuscular plasmid VEGF with some beneficial effects in diabetic patients with critical limb ischemia, together with the novel mechanistic insights from the mouse studies, may provide a handhold for the development of new trial protocols. Finally, progress in the research field of arteriogenesis may not only prove beneficial for the treatment of peripheral artery disease, which is the aim of this dissertation, but also of coronary heart disease, the leading cause of death to date. One of many challenges is to elucidate similarities and differences in cellular and molecular mechanisms of vascular growth between limb and heart, which may help to extrapolate results from clinical trials conducted in the field of vascular surgery towards the field of cardiology and vice versa.This work was financially supported by the TNO-LUMC-VUMC tripartite angiogenesis program. Financial support by the Netherlands Heart Foundation and TNO-Quality of Life for the publication of this thesis is gratefully acknowledged.UBL - phd migration 201

Reading on paper and screens: advantages, disadvantages, and digital divide

34-43The paper studies reading in the digital age and grounds characteristic reading features on paper and the screen. We studied various scientific points on the uniqueness of reading activities in various formats, effectiveness, and perception of readers about printed and electronic texts. The study concludes that screen-reading practices keep evolving. Digital reading dynamically changes the communication environment, speeding up and simplifying access to information. The new reading format has many opportunities and advantages, and it transforms the reading skills and habits of society. Although it revives social reading, it intensifies the digital divide, leading to a secondary mental cognitive digital divide, and modifies the personal reading experience of contemporaries

Managing the complexity of doing it all : an exploratory study on students' experiences when trained stepwise in conducting consultations

Background: At most medical schools the components required to conduct a consultation, medical knowledge, communication, clinical reasoning and physical examination skills, are trained separately. Afterwards, all the knowledge and skills students acquired must be integrated into complete consultations, an art that lies at the heart of the medical profession. Inevitably, students experience conducting consultations as complex and challenging. Literature emphasizes the importance of three didactic course principles: moving from partial tasks to whole task learning, diminishing supervisors' support and gradually increasing students' responsibility. This study explores students' experiences of an integrated consultation course using these three didactic principles to support them in this difficult task. Methods: Six focus groups were conducted with 20 pre-clerkship and 19 clerkship students in total. Discussions were audiotaped, transcribed and analysed by Nvivo using the constant comparative strategy within a thematic analysis. Results: Conducting complete consultations motivated students in their learning process as future physician. Initially, students were very much focused on medical problem solving. Completing the whole task of a consultation obligated them to transfer their theoretical medical knowledge into applicable clinical knowledge on the spot. Furthermore, diminishing the support of a supervisor triggered students to reflect on their own actions but contrasted with their increased appreciation of critical feedback. Increasing students' responsibility stimulated their active learning but made some students feel overloaded. These students were anxious to miss patient information or not being able to take the right decisions or to answer patients' questions, which sometimes resulted in evasive coping techniques, such as talking faster to prevent the patient asking questions. Conclusion: The complex task of conducting complete consultations should be implemented early within medical curricula because students need time to organize their medical knowledge into applicable clinical knowledge. An integrated consultation course should comprise a step-by-step teaching strategy with a variety of supervisors' feedback modi, adapted to students' competence. Finally, students should be guided in formulating achievable standards to prevent them from feeling overloaded in practicing complete consultations with simulated or real patients

Readiness of the Belgian network of sentinel general practitioners to deliver electronic health record data for surveillance purposes: results of survey study

<p>Abstract</p> <p>Background</p> <p>In order to proceed from a paper based registration to a surveillance system that is based on extraction of electronic health records (EHR), knowledge is needed on the number and representativeness of sentinel GPs using a government-certified EHR system and the quality of EHR data for research, expressed in the compliance rate with three criteria: recording of home visits, use of prescription module and diagnostic subject headings.</p> <p>Methods</p> <p>Data were collected by annual postal surveys between 2005 and 2009 among all sentinel GPs. We tested relations between four key GP characteristics (age, gender, language community, practice organisation) and use of a certified EHR system by multivariable logistic regression. The relation between EHR software package, GP characteristics and compliance with three quality criteria was equally measured by multivariable logistic regression.</p> <p>Results</p> <p>A response rate of 99% was obtained. Of 221 sentinel GPs, 55% participated in the surveillance without interruption from 2005 onwards, i.e. all five years, and 78% were participants in 2009. Sixteen certified EHR systems were used among 91% of the Dutch and 63% of the French speaking sentinel GPs. The EHR software package was strongly related to the community and only one EHR system was used by a comparable number of sentinel GPs in both communities. Overall, the prescription module was always used and home visits were usually recorded. Uniform subject headings were only sometimes used and the compliance with this quality criterion was almost exclusively related to the EHR software package in use.</p> <p>Conclusions</p> <p>The challenge is to progress towards a sentinel network of GPs delivering care-based data that are (partly) extracted from well performing EHR systems and still representative for Belgian general practice.</p

Eye–hand coordination during manual object transport with the affected and less affected hand in adolescents with hemiparetic cerebral palsy

In the present study we investigated eye–hand coordination in adolescents with hemiparetic cerebral palsy (CP) and neurologically healthy controls. Using an object prehension and transport task, we addressed two hypotheses, motivated by the question whether early brain damage and the ensuing limitations of motor activity lead to general and/or effector-specific effects in visuomotor control of manual actions. We hypothesized that individuals with hemiparetic CP would more closely visually monitor actions with their affected hand, compared to both their less affected hand and to control participants without a sensorimotor impairment. A second, more speculative hypothesis was that, in relation to previously established deficits in prospective action control in individuals with hemiparetic CP, gaze patterns might be less anticipatory in general, also during actions performed with the less affected hand. Analysis of the gaze and hand movement data revealed the increased visual monitoring of participants with CP when using their affected hand at the beginning as well as during object transport. In contrast, no general deficit in anticipatory gaze control in the participants with hemiparetic CP could be observed. Collectively, these findings are the first to directly show that individuals with hemiparetic CP adapt eye–hand coordination to the specific constraints of the moving limb, presumably to compensate for sensorimotor deficits

Identification of the initial molecular changes in response to circulating angiogenic cells-mediated therapy in critical limb ischemia

BackgroundCritical limb ischemia (CLI) constitutes the most aggressive form of peripheral arterial occlusive disease, characterized by the blockade of arteries supplying blood to the lower extremities, significantly diminishing oxygen and nutrient supply. CLI patients usually undergo amputation of fingers, feet, or extremities, with a high risk of mortality due to associated comorbidities.Circulating angiogenic cells (CACs), also known as early endothelial progenitor cells, constitute promising candidates for cell therapy in CLI due to their assigned vascular regenerative properties. Preclinical and clinical assays with CACs have shown promising results. A better understanding of how these cells participate in vascular regeneration would significantly help to potentiate their role in revascularization.Herein, we analyzed the initial molecular mechanisms triggered by human CACs after being administered to a murine model of CLI, in order to understand how these cells promote angiogenesis within the ischemic tissues.MethodsBalb-c nude mice (n:24) were distributed in four different groups: healthy controls (C, n:4), shams (SH, n:4), and ischemic mice (after femoral ligation) that received either 50 mu l physiological serum (SC, n:8) or 5x10(5) human CACs (SE, n:8). Ischemic mice were sacrificed on days 2 and 4 (n:4/group/day), and immunohistochemistry assays and qPCR amplification of Alu-human-specific sequences were carried out for cell detection and vascular density measurements. Additionally, a label-free MS-based quantitative approach was performed to identify protein changes related.ResultsAdministration of CACs induced in the ischemic tissues an increase in the number of blood vessels as well as the diameter size compared to ischemic, non-treated mice, although the number of CACs decreased within time. The initial protein changes taking place in response to ischemia and more importantly, right after administration of CACs to CLI mice, are shown.ConclusionsOur results indicate that CACs migrate to the injured area; moreover, they trigger protein changes correlated with cell migration, cell death, angiogenesis, and arteriogenesis in the host. These changes indicate that CACs promote from the beginning an increase in the number of vessels as well as the development of an appropriate vascular network.Institute of Health Carlos III, ISCIII; Junta de Andaluci

Perspectives on key principles of generalist medical practice in public service in sub-saharan africa: a qualitative study

The original publication is available at http://www.biomedcentral.comAbstract Background: The principles and practice of Family Medicine that arose in developed Western countries have been imported and adopted in African countries without adequate consideration of their relevance and appropriateness to the African context. In this study we attempted to elicit a priori principles of generalist medical practice from the experience of long-serving medical officers in a variety of African counties, through which we explored emergent principles of Family Medicine in our own context. Methods A descriptive study design was utilized, using qualitative methods. 16 respondents who were clinically active medical practitioners, working as generalists in the public services or non-profit sector for at least 5 years, and who had had no previous formal training or involvement in academic Family Medicine, were purposively selected in 8 different countries in southern, western and east Africa, and interviewed. Results The respondents highlighted a number of key issues with respect to the external environment within which they work, their collective roles, activities and behaviours, as well as the personal values and beliefs that motivate their behaviour. The context is characterized by resource constraints, high workload, traditional health beliefs, and the difficulty of referring patients to the next level of care. Generalist clinicians in sub-Saharan Africa need to be competent across a wide range of clinical disciplines and procedural skills at the level of the district hospital and clinic, in both chronic and emergency care. They need to understand the patient's perspective and context, empowering the patient and building an effective doctor-patient relationship. They are also managers, focused on coordinating and improving the quality of clinical care through teamwork, training and mentoring other health workers in the generalist setting, while being life-long learners themselves. However, their role in the community, was found to be more aspirational than real. Conclusions The study derived a set of principles for the practice of generalist doctors in sub-Saharan Africa based on the reported activities and approaches of the respondents. Patient-centred care using a biopsychosocial approach remains as a common core principle despite wide variations in context. Procedural and hospital care demands a higher level of skills particularly in rural areas, and a community orientation is desirable, but not widely practiced. The results have implications for the postgraduate training of family physicians in sub-Saharan Africa, and highlight questions regarding the realization of community-orientated primary care.Publishers' Versio

Reviewing, indicating, and counting books for modern research evaluation systems

In this chapter, we focus on the specialists who have helped to improve the conditions for book assessments in research evaluation exercises, with empirically based data and insights supporting their greater integration. Our review highlights the research carried out by four types of expert communities, referred to as the monitors, the subject classifiers, the indexers and the indicator constructionists. Many challenges lie ahead for scholars affiliated with these communities, particularly the latter three. By acknowledging their unique, yet interrelated roles, we show where the greatest potential is for both quantitative and qualitative indicator advancements in book-inclusive evaluation systems.Comment: Forthcoming in Glanzel, W., Moed, H.F., Schmoch U., Thelwall, M. (2018). Springer Handbook of Science and Technology Indicators. Springer Some corrections made in subsection 'Publisher prestige or quality