Dissertationen zum Thema „Neovascularisation“

The aim of this project was to develop in vitro models for angiogenesis that have the capability of combining pro-angiogenic cells with an extracellular matrix (ECM) component that can be monitored under flow conditions to learn more about the ‘rules’ of angiogenesis. Significant advancement has been made in the field of tissue engineering in recent years, however one of the current obstacles limiting progression is the production of thick, complex tissues due to the lack of rapid neovascularisation of the constructs upon implantation. Blood vessel formation is tightly regulated and relies on the chronologically precise adjustment of vessel growth, maturation and suppression of endothelial cell growth - all of which are controlled by a large number of factors which influence each other. To induce vascularisation within tissue-engineered (TE) substitutes the same processes need to occur. A number of different vascularisation strategies have been investigated in an attempt to overcome this issue but as yet there is no unified solution to this problem. The most promising attempts have used scaffolds with vascular architectures, perfusion conditions and relevant cell types. Although it is recognised that perfusion conditions, the cell type and scaffold architecture are important with regards to vascularisation strategies many of the techniques fail to consider them in combination. It is therefore important to take a step back and understand how these factors work together to i promote angiogenesis in order to advance this crucial area. This lack of understanding is further compounded by the deficiencies of current angiogenesis models. Current in vitro models fail to combine the use of supporting cells, the extracellular matrix and fluid flow in 3D. Although this complexity exists within in vivo models such assays are primarily limited by the species used, organ sites available and complicated analysis techniques. In this project two in vitro angiogenesis models were developed. The first was derived from the decellularisation of a rat jejunum. Characterisation showed the retention of key extracellular matrix (ECM) components and the removal of almost all cellular material. Re-endothelialisation with human dermal endothelial cells (HDMECs) of the patent vascular network showed enhanced results when co-cultured with human dermal fibroblasts (HDFs). In an attempt to induce angiogenesis, vascular endothelial growth factor (VEGF) loaded gels were placed on top of the scaffold whilst being continuously perfused with media. Placing VEGF loaded gels onto the recellularised jejunum led to the expression of the Notch ligand Delta- like-4 (DLL4) by HDMECs indicating their transformation into tip cells which are synonymous with sprouting angiogenesis. The second was produced through the combination of robocasting and electrospinning. Nanofibrous poly(3-hydroxybutyrate-co-3- hydroxyvalerate) (PHBV) scaffolds with hollow channels capable of perfusion were produced that could be re-endothlialised with HDMECs. Again the addition of HDFs enhanced cellular distribution in the channels. Placing VEGF loaded gels onto the surface of the scaffolds led to the outgrowth of HDMECs into the gel, forming perfusable tubules. Overall these two models overcome limitations of current in vitro models since they offer the capability of combining pro-angiogenic cells with ECM components that can be monitored under flow conditions. With further development they could provide more sophisticated platforms upon which to investigate the angiogenic process.

[Truncated abstract] One of the barriers to understanding and preventing proliferative diabetic retinopathy in humans has been the lack of an appropriate animal model. Historically dog, rat and mouse models of diabetic retinopathy have been studied but none of these exhibit the later changes of proliferative diabetic retinopathy. Animals can be rendered diabetic by surgical pancreatectomy or the use of chemicals such as allozan or streptozotocin or by feeding of a high galactose diet. Alternatively, spontaneous rodent models of diabetes have been examined such as the BB rat, KK mouse or NOD mouse. However, in each case the retinal vascular changes observed are those of early nonproliferative diabetic retinopathy comprising at most saccular microaneurysms, increased thickness of the capillary basement membrane, acellular capillaries and pericyte ghosts. … Fluorecein angiography of this transgenic line clearly demonstrates the presence of leaky new vessels, by the appearance of leakage spots scattered throughout the retina from 1 month of age. These mice constitute a valuable model of diabetic retinopathy. Neovascularization in this animal model is induced by VEGF as in human diabetic retinopathy. The source of VEGF in human diabetic retinopathy is the ischemic inner retina. In this transgenic model the source of VEGF are the photoreceptor cells, which are situated just underneath the inner retina. The neovascularization is not dependent on a particular developmental stage and there is no spontaneous regression of new vessels. Thus any results generated in this model are highly relevant to human diabetic retinopathy.

Age-related macular degeneration (AMD) is the major cause of vision loss in the developed world. AMD is a chronic progressive disorder of the outer retina leading to vision loss from atrophy (geographic atrophy) and/or the development of choroidal neovascularisation (CNV). Mounting evidence indicates the importance of innate immunity in its pathogenesis. This thesis describes a programme of work conducted with the aim of further understanding the role of innate immune cells in AMD. Analysis of mouse cell suspensions by flow cytometry demonstrated significantly greater densities of innate immune cell populations in the RPE-choroid than in the neurosensory retina. Dendritic cells accumulated in both tissues with increasing age, and this process was accelerated in mice deficient in a chemokine upregulated in the aged choroid - CCL2. Innate immune cells were recruited to CNV lesions in mouse models of laser-induced and spontaneous CNV. Increasing age was found to correlate with the extent of laser CNV lesion size but not with recruitment of innate immune cells. Impaired recruitment of innate immune cells in CCL2-deficient mice was associated with a smaller laser-CNV lesion size, which nonetheless increased with age. Attenuation of CNV lesion size by targeting key angiogenic pathways using the small molecule pazopanib or by direct targeting of innate immune cells by induction of alternative activation using a CD200R agonist was associated with subtle increases in the recruitment of innate immune cell subpopulations to CNV lesions. Findings did not support the hypothesis that age-related vulnerability to laser CNV is a consequence of age-related changes in innate immune cell populations. However, the results indicated that CCL2 controls dendritic cell migration in the ageing retina and the recruitment of innate immune cells to CNV lesions. Further investigation of these pathways may lead to better treatments in the prevention and management of AMD.

Atherosclerosis is a chronic inflammatory disorder, initiated by arterial wall injury, mediated by well-recognised cardiovascular risk factors and culminating in formation of plaques, the patho-biological substrate that precedes events such as stroke and myocardial infarction. Intraplaque neovascularisation (IPN) is one of several defence mechanisms in response to atherosclerosis. With development of an atherosclerotic plaque within the intima, the distance between the deeper intimal layers and the luminal surface increases, producing hypoxia within the arterial wall. This stimulates release of pro-angiogenic factors that induces neoangiogenesis in an attempt to normalise oxygen tension. However, these neo-vessels are fragile, immature and leaky and thought to be the primary cause of intraplaque haemorrhage, now appreciated to be a key risk factor for plaque rupture. Therefore, the presence of IPN is now widely recognised as a precursor of the “vulnerable plaque”. Contrast-enhanced ultrasound (CEUS) is a non-invasive method of imaging carotid plaques and, as contrast bubbles travel wherever erythrocytes travel, they permit visualization of IPN. Prior research studies have demonstrated that CEUS can detect IPN with a high degree of accuracy (on comparison with histological plaque specimens) and have shown a relationship between extent of plaque neovessels and plaque echogenicity and between plaque neovascularization and prior cardiovascular events. However, CEUS is a relatively recently described imaging technique and there were a number of unanswered questions in this field, some of which formed the basis for study in this research Thesis. In this Thesis, research studies were conducted on human subjects using CEUS imaging to identify IPN and its determinants. The incidence and determinants of IPN in healthy asymptomatic individuals was unknown and was studied in subjects from the London Life Sciences Population (LOLIPOP) study, a large study exploring mechanisms for differences in cardiovascular disease (CVD) between South Asian and European White individuals. The study found that approximately half of all plaques contain IPN. The only variable associated with IPN presence in an adjusted analysis was Asian ethnicity. This finding potentially has significant implications as it may help explain, in part, the greater CVD burden observed in Asian populations. A study comparing visualization of the carotid tree during B-mode and CEUS imaging was also conducted. Both IMT visualization and plaque detection were significantly improved by CEUS, implying that CEUS is superior to B-mode imaging for detection of sub-clinical atherosclerosis. Radiotherapy (RT) damages arterial walls and promotes atherosclerosis. The carotid arteries frequently receive significant incidental doses of radiation during RT treatment of head and neck cancers. The effect of RT on plaque composition – specifically IPN – had not been studied and thus a collaborative cardio-oncological study was conducted to assess the effects of RT upon IPN in cancer survivors who had previously received RT. A significant association between RT and IPN was found which may provide insights into the mechanisms underlying the increased stroke risk amongst cancer survivors treated by RT. Finally, a collaboration with biophysicists was formed to develop and validate a novel algorithm for quantitative analysis of IPN. Patients clinically scheduled to undergo carotid endarterectomy were recruited and underwent CEUS imaging prior to surgery. This study did not achieve its principal aims due to challenges with patient recruitment, challenges in image quality and with the quantification software also. Future directions of study in this promising field have been addressed in the thesis summary.

The principal aim of the present thesis was to prospectively follow (clinical status and ultrasound + Doppler findings) the patellar tendons in the young elite volleyball players at the Swedish National Centre for high school volleyball in Falköping. In an Olympic weightlifter with chronic painful jumper´s knee, the effects of treatment with sclerosing injections followed by early instituted very heavy weightlifting training, was also evaluated. First, in a prevalence study, we demonstrated that the clinical diagnosis patellar tendinopathy-jumper’s knee, together with structural tendon changes and vascularisation in the painful area of the tendon, was demonstrated in 12/114 tendons in Swedish junior elite volleyball players, but not in any tendons of individually matched (age, height and weight) not regularly sports active controls. Structural tendon changes alone was demonstrated among the volleyball players but also among the controls. In a 7 months prospective study of a total of 120 tendons, we demonstrated that the clinical diagnosis patellar tendinopathy-jumper’s knee was associated with neovessels/vascularity in the area with structural tendon changes in 17/19 tendons. Seventy tendons that at start were clinically normal, and had normal ultrasound + Doppler findings, remained clinically normal after 7 months with intensive training and playing volleyball. In a 3-year prospective study it was demonstrated that normal clinical tests and normal ultrasound + Doppler findings at school start, indicated a low risk (8%) for these players to sustain patellar tendinopathy-jumper’s knee during the 3 school years with intensive training and playing. In a case study, involving an Olympic elite weightlifter with chronic painful patellar tendinopathy-jumper’s knee, successful treatment with ultrasound and Doppler-guided injection of the sclerosing agent polidocanol, allowed for pain-free very heavy weight training two weeks after treatment. Further heavy weightlifting training on a daily basis, preparing for European Championships, was done without causing tendon rupture and/or pain. Key words: Jumper’s knee, Patellar tendinopathy, Chronic pain, Ultrasonography, Doppler, Neovascularisation, Volleyball, Weightlifting

Ischaemic heart disease (IHD) is the leading cause of death worldwide. Currently, even optimal medical therapies do not attenuate deterioration of the left ventricular (LV) function completely. Stem cell therapies, and recently cardiac stem cell therapies, have emerged as potential novel treatments for IHD. However, clinical evidence from randomised controlled studies has shown mixed results. Thus understanding what patient-related factors may affect the therapeutic performance of the cells may help improving treatment outcomes. The studies described in this thesis aim to understand how cardiac progenitor cells (CPCs) can re-vascularise ischaemic myocardium and promote functional repair of the heart. Resident CPCs were isolated and expanded from the right atrial appendage of 68 patients following the ‘cardiosphere’ method (cardiosphere-derived cells or CDCs). They resemble mesenchymal progenitors as they lack the expression of endothelial and haematopoietic cell surface markers but express mesenchymal progenitor cell markers (e.g. CD105, CD90). Cell function was evaluated by support of angiogenesis, mesenchymal lineage differentiation potential in vitro, and improvement in heart function in vivo. Notably in vitro, CDC from different patients differed in their angiogenic supportive and differentiation potentials. In a rodent model of myocardial infarction (MI), transplantation of CDC reduced infarct size significantly (p<0.05). However, only those CDCs with a robust pro-angiogenic ability in vitro improved vessel density and heart systolic function (p<0.05) in vivo. A multiple regression model, which accounted for 51% of the variability observed, identified New York Heart Association (NYHA) class, smoking, hypertension, type of ischaemic disease and diseased vessel as independent predictors of angiogenesis. In addition, gene expression analyses revealed that differential gene expression of several extracellular matrix components (e.g. CUX1, COL1A2, BMP1 genes and microRNA-29b) could explain the differences observed in CDC’s vascular supportive function. In summary, this is the first description of variability in the pro-angiogenic and differentiation potential of CDCs and its correlation with their therapeutic potential. This study indicates that patient stratification may need to be included in the design of future trials to improve the efficacy of cell-based therapies.

Doctor of Philosophy (PhD)<br>BACKGROUND: The accumulation of abnormal extracellular deposits beneath the retinal pigment epithelium characterises the pathology of early age-related macular degeneration. However, the histopathological threshold at which age-related changes become early AMD is not defined, and the effect of each of the deposits (basal laminar deposit and membranous debris) on disease progression is poorly understood. Evidence suggests that macrophages play a key role in the development of AMD lesions, but the influence of basal laminar deposit (BLamD) and membranous debris on the recruitment and programming of local macrophages has not been explored. Although evidence also suggests that inflammation and innate immunity are involved in AMD, the significance of anti-retinal autoantibodies to disesase pathogenesis is not known. AIMS: (i) To determine the histopathological threshold that distinguishes normal ageing from early AMD; (ii) to determine the influence of BLamD and membranous debris on disease progression; (iii) to examine whether distinct early AMD phenotypes exist based on clinicopathological evidence; (iv) to determine the histopathological context in which Bruch’s membrane macrophages first found; (v) to examine the relationship between Bruch’s membrane macrophages and subclinical neovascularisation; (vi) to determine if the progressive accumulation of BLamD and membranous debris alters the immunophenotype of Bruch’s membrane macrophages and/or resident choroidal macrophages; (vii) to determine if the anti-retinal autoantibody profile differs significantly between normal individuals and those with early AMD, neovascular AMD or geographic atrophy; (viii) to examine whether baseline anti-retinal autoantibodies can predict progression to advanced AMD in individuals with early AMD; and (ix) to examine whether baseline anti-retinal autoantibodies can predict vision loss in individuals with neovascular AMD. METHODS:Clinicopathological studies were performed to correlate progressive accumulation of BLamD and membranous debris to fundus characteristics and visual acuity, as well as to sub-macular Bruch’s membrane macrophage count. Immunohistochemical studies were perfomed to determine whether the presence of BLamD and membranous debris altered the programming of Bruch’s membrane or resident choroidal macrophages. The presence of serum anti-retinal autoantibodies was determined by western blotting, and the association with disease progression examined in early and neovascular AMD. RESULTS: The presence of both basal linear deposit (BLinD) and a continuous layer of BLamD represents threshold early AMD histopathologically, which was seen clinically as a normal fundus in the majority of cases. Membranous debris accumulation appeared to influence the pathway of progression from early AMD to advanced AMD. Bruch’s membrane macrophages were first noted when a continuous layer of BLamD and clinical evidence of early AMD were present, and increased with the amount of membranous debris in eyes with thin BLamD. Eyes with subclinical CNV had high macrophage counts and there was some evidence of altered resident choroidal macrophage programming in the presence of BLamD and membranous debris. Serum anti-retinal autoantibodies were found in a higher proportion of early AMD participants compared with both controls and participants with neovascular AMD, and in a higher proportion of individuals with atrophic AMD compared to those with neovascular AMD. The presence of baseline anti-retinal autoantibodies in participants with early AMD was not associated with progression to advanced AMD. Participants with neovascular AMD lost more vision over 24 months if they had IgG autoantibodies at baseline compared to autoantibody negative participants. CONCLUSIONS: The finding that eyes with threshold early AMD appear clinically normal underscores the need to utilise more sophisticated tests to enable earlier disease detection. Clinicopathological evidence suggests two distinct early AMD phenotypes, which follow two pathways of AMD progression. Macrophage recruitment and programming may be altered by the presence of BLamD and membranous debris, highlighting the need to further characterise the biology of human resident choroidal macropahges. Anti-retinal autoantibodies can be found in both control and AMD sera, and future approaches that allow the examination of subtle changes in complex repertoires will determine whether they are involved in AMD disease pathogenesis.

Malgre un investissement considerable, les resultats obtenus pour l'instant par la therapie genique sont encore tres modestes, principalement du fait de problemes de vectorisation. L'objectif de ce travail etait double : d'une part ameliorer les vecteurs utilises pour permettre le transfert de gene aux cellules souches hematopoietiques (csh) et, d'autre part, evaluer le potentiel antitumoral de la secretion systemique d'une molecule antiangiogenique naturelle a partir du systeme hematopoietique. Nous decrivons tout d'abord une technique originale de transduction retrovirale des cellules cd34+ humaines apres infection par un vecteur aav apportant l'adn complementaire du recepteur ecotrope. Ensuite, nous decrivons une serie de vecteurs permettant de transferer dans les csh un gene d'interet associe au gene de la green fluorescent protein (gfp) dans de bonnes conditions de titre et de stabilite. La selection par cytofluorimetrie de flux des csh transduites par ces vecteurs avant leur reinjection nous a permis d'obtenir des resultats remarquables en terme de taux de cellules hematopoietiques peripheriques finalement transformees et, exprimant le transgene a long terme. Nous decrivons leur application au transfert du gene de la -globine humaine chez la souris et a un modele preclinique de protoporphyrie hereditaire. Utilisant ces techniques avec un adnc codant pour une forme secretee de l'endostatine, nous avons obtenu, a long terme, des taux circulants de cette molecule 7 fois superieurs a la normale, sans toutefois observer d'effet antitumoral notable. Ces resultats constituent un net progres dans le cadre de la therapie genique appliquee au systeme hematopoietique. Pour ce qui est du controle de la neoangiogenese, nos resultats mettent en cause l'efficacite reelle de l'endostatine.

The aberrant growth of blood vessels in the posterior segment of the eye is a major symptom of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world. This project has enabled a significant step forward in the study of blood vessels that become diseased in patients suffering from AMD. In doing so, a previously uncharacterised cell type was identified, and with further refinement, could lead to potential novel strategies for wet-AMD therapies.

BACKGROUND: The accumulation of abnormal extracellular deposits beneath the retinal pigment epithelium characterises the pathology of early age-related macular degeneration. However, the histopathological threshold at which age-related changes become early AMD is not defined, and the effect of each of the deposits (basal laminar deposit and membranous debris) on disease progression is poorly understood. Evidence suggests that macrophages play a key role in the development of AMD lesions, but the influence of basal laminar deposit (BLamD) and membranous debris on the recruitment and programming of local macrophages has not been explored. Although evidence also suggests that inflammation and innate immunity are involved in AMD, the significance of anti-retinal autoantibodies to disesase pathogenesis is not known. AIMS: (i) To determine the histopathological threshold that distinguishes normal ageing from early AMD; (ii) to determine the influence of BLamD and membranous debris on disease progression; (iii) to examine whether distinct early AMD phenotypes exist based on clinicopathological evidence; (iv) to determine the histopathological context in which Bruch’s membrane macrophages first found; (v) to examine the relationship between Bruch’s membrane macrophages and subclinical neovascularisation; (vi) to determine if the progressive accumulation of BLamD and membranous debris alters the immunophenotype of Bruch’s membrane macrophages and/or resident choroidal macrophages; (vii) to determine if the anti-retinal autoantibody profile differs significantly between normal individuals and those with early AMD, neovascular AMD or geographic atrophy; (viii) to examine whether baseline anti-retinal autoantibodies can predict progression to advanced AMD in individuals with early AMD; and (ix) to examine whether baseline anti-retinal autoantibodies can predict vision loss in individuals with neovascular AMD. METHODS:Clinicopathological studies were performed to correlate progressive accumulation of BLamD and membranous debris to fundus characteristics and visual acuity, as well as to sub-macular Bruch’s membrane macrophage count. Immunohistochemical studies were perfomed to determine whether the presence of BLamD and membranous debris altered the programming of Bruch’s membrane or resident choroidal macrophages. The presence of serum anti-retinal autoantibodies was determined by western blotting, and the association with disease progression examined in early and neovascular AMD. RESULTS: The presence of both basal linear deposit (BLinD) and a continuous layer of BLamD represents threshold early AMD histopathologically, which was seen clinically as a normal fundus in the majority of cases. Membranous debris accumulation appeared to influence the pathway of progression from early AMD to advanced AMD. Bruch’s membrane macrophages were first noted when a continuous layer of BLamD and clinical evidence of early AMD were present, and increased with the amount of membranous debris in eyes with thin BLamD. Eyes with subclinical CNV had high macrophage counts and there was some evidence of altered resident choroidal macrophage programming in the presence of BLamD and membranous debris. Serum anti-retinal autoantibodies were found in a higher proportion of early AMD participants compared with both controls and participants with neovascular AMD, and in a higher proportion of individuals with atrophic AMD compared to those with neovascular AMD. The presence of baseline anti-retinal autoantibodies in participants with early AMD was not associated with progression to advanced AMD. Participants with neovascular AMD lost more vision over 24 months if they had IgG autoantibodies at baseline compared to autoantibody negative participants. CONCLUSIONS: The finding that eyes with threshold early AMD appear clinically normal underscores the need to utilise more sophisticated tests to enable earlier disease detection. Clinicopathological evidence suggests two distinct early AMD phenotypes, which follow two pathways of AMD progression. Macrophage recruitment and programming may be altered by the presence of BLamD and membranous debris, highlighting the need to further characterise the biology of human resident choroidal macropahges. Anti-retinal autoantibodies can be found in both control and AMD sera, and future approaches that allow the examination of subtle changes in complex repertoires will determine whether they are involved in AMD disease pathogenesis.

Les maladies cardiovasculaires, principalement causées par l'athérosclérose, demeurent la première cause de mortalité dans le monde. L'athérosclérose est une maladie inflammatoire chronique de la paroi vasculaire provoquée par l'internalisation de lipides dans l'espace sous-endothéliale. Cette accumulation anormale déclenche l'infiltration de leucocytes et l'activation des cellules musculaires lisses (CML). Plus que l'obstruction des vaisseaux, l'instabilité et la rupture des plaques sont considérées comme les événements les plus délétères à l'origine d'infarctus du myocarde par exemple. Les deux principaux processus responsables de l'instabilité des plaques sont la calcification vasculaire et la néovascularisation intraplaque. La calcification vasculaire consiste en une minéralisation de la plaque d'athérosclérose sous forme de micro-calcifications et de macro-calcifications. Si cette dernière a été considérée comme stabilisatrice, elle diminue aussi l'élasticité des vaisseaux contribuant à l'hypertension, à la sténose aortique et à la vulnérabilité des plaques. Quant à la néovascularisation intraplaque, elle résulte de la formation de zones hypoxiques intraplaques qui provoque la formation de néovaisseaux immatures et perméables. L'angiogenèse intraplaque favorise la progression des plaques et accroît le risque de rupture. Nous cherchons à identifier ici les mécanismes responsables de ces deux phénomènes. La compréhension des mécanismes sous-jacents pourrait en effet ouvrir la voie à de nouvelles cibles thérapeutiques.Grâce à une approche multi-omics, nous avons identifié le récepteur nucléaire Rev-erbα comme un facteur clé particulièrement dérégulé dans les cellules de patients diabétiques ayant un haut risque cardiovasculaire comparé aux cellules de patients diabétiques avec un bas risque cardiovasculaire. Rev-erbα est un inhibiteur de la transcription, activé par des ligands naturels ou synthétiques, impliqué dans la régulation du métabolisme lipidique et de la réponse inflammatoire. Par ailleurs, des expériences de microdissection laser réalisées sur des endartériectomies humaines montrent que l'expression de REV-ERBα est plus faible dans les zones calcifiées et vascularisées suggérant un rôle protecteur de REV-ERBα.Dans un modèle de souris LDLr-/- âgées de 18 mois, la délétion de Rev-erbα accélère la progression et la complexité de la plaque. Par ailleurs, une analyse transcriptomique menée sur les aortes de ces souris révèle que les gènes associés aux voies de la calcification vasculaire et de l'angiogenèse intraplaque sont particulièrement enrichis lorsque Rev-erbα est délété suggérant que Rev-erbα protégerait contre l'athérosclérose, la calcification vasculaire et la néovascularisation.La déficience en Rev-erbα favorise en effet le développement de l'athérosclérose, augmente le dépôt de calcium et induit la différenciation ostéoblastique des CML. Cet effet sur la différenciation ostéoblastique des CML est amplifié en présence de cytokines pro-inflammatoires in vitro. Des analyses transcriptomiques réalisées sur CML en cours de différentiation nous ont permis d'identifier la hyaluronidase Cemip comme facteur important dans le rôle que joue Rev-erbα sur la différenciation ostéoblastique.Par ailleurs, l'analyse d'artères brachiocéphaliques par 3DISCO montre que l'absence de Rev-erbα favorise le développement d'un réseau vasculaire intraplaque plus complexe et immature que chez les souris contrôles. Au niveau moléculaire, il semblerait que Rev-erbα contrôle l'expression et de la sécrétion de facteurs pro-angiogéniques de façon cellule-spécifique.En conclusion, Rev-erbα apparaît comme un régulateur clé de la calcification vasculaire et de la sécrétion des facteurs pro-angiogéniques par les cellules de la plaque. Ces résultats permettent d'identifier Rev-erbα comme une nouvelle cible thérapeutique prometteuse pour réduire le risque cardiovasculaire résiduel chez les patients à très haut risque cardiovasculaire<br>Cardiovascular disease, mainly caused by atherosclerosis, remains the leading cause of death worldwide. Atherosclerosis is a chronic inflammatory disease of the vascular wall caused by the internalization of lipids into the subendothelial space. This abnormal accumulation triggers leukocyte infiltration and smooth muscle cell (SMC) activation. More than vascular occlusion, plaque instability and rupture are considered to be the most deleterious events at the origin of myocardial infarction, for exemple. The two main processes responsible for plaque instability are vascular calcification and intraplaque neovascularization. Vascular calcification is the mineralization of atherosclerotic plaque in the form of micro-calcifications and macro-calcifications. While the latter is considered stabilizing, it also reduces vascular elasticity and contributes to hypertension, aortic stenosis, and plaque vulnerability. Intraplaque neovascularization results from the formation of intraplaque hypoxic zones, leading to the formation of immature, permeable neovessels. Intraplaque angiogenesis promotes plaque progression and increases the risk of rupture. Our aim is to identify the mechanisms responsible for these two phenomena. Understanding the underlying mechanisms may provide new therapeutic targets.Using a multi-omics approach, we identified the nuclear receptor Rev-erbα as a key factor deregulated in cells from diabetic patients at high cardiovascular risk compared to cells from diabetic patients at low cardiovascular risk. Rev-erbα is a natural or synthetic ligand-activated transcription inhibitor involved in the regulation of lipid metabolism and inflammatory response. Furthermore, laser microdissection experiments performed on human endarterectomies show that REV-ERBα expression is lower in calcified and vascularized areas, suggesting a protective role for REV-ERBα.In an 18-month-old LDLr-/- mouse model, deletion of Rev-erbα accelerates plaque progression and complexity. Furthermore, transcriptomic analysis of aortas from these mice revealed that genes associated with vascular calcification and intraplaque angiogenesis pathways are enriched when Rev-erbα is deleted, suggesting that Rev-erbα protects against atherosclerosis, vascular calcification and neovascularization. Rev-erbα deficiency promotes the development of atherosclerosis, increases calcium deposition and induces osteoblastic differentiation of SMC. This effect on osteoblastic differentiation of SMC is enhanced in the presence of pro-inflammatory cytokines in vitro. Transcriptomic analyses performed on differentiating SMC allowed us to identify the hyaluronidase Cemip as an important factor in the role of Rev-erbα in osteoblastic differentiation. Furthermore, 3DISCO analysis of brachiocephalic arteries shows that the absence of Rev-erbα promotes the development of a more complex and immature intraplaque vascular network than in control mice. At the molecular level, Rev-erbα appears to control the expression and secretion of pro-angiogenic factors in a cell-specific manner.In conclusion, Rev-erbα appears to be a key regulator of vascular calcification and secretion of pro-angiogenic factors by plaque cells. These results identify Rev-erbα as a promising new therapeutic target for reducing residual cardiovascular risk in patients at very high cardiovascular risk

Trabalho final de mestrado integrado em Medicina (Oftalmologia), apresentado à Faculdade de Medicina da Universidade de Coimbra<br>Objectives To characterize the progression of eye fundus changes by multimodal imaging and to identify morphological and/or functional predictors of conversion in wet AMD in the fellow eyes of patients with exudative AMD. Sample Description and Methods Single-center, prospective, observational, longitudinal 2-year study. Sixty-two patients were enrolled in the study, with diagnosis of neovascular age-related macular degeneration in one eye and age-related maculopathy in the fellow eye. Each patient underwent a detailed ocular and medical history, a complete ophthalmologic examination with color fundus photography, fluorescein angiography, indocyanine green angiography and fundus autofluorescence imaging at baseline and repeated at six-month intervals. CFP images were graded resorting to the RetmarkerAMD (Critical Health SA) software, a computer assisted grading system based on the International Classification Grading System guidelines (Bird, Bressler et al., 1995). Results Fifty-three patients completed the two-year follow-up period, eighteen of which converted to the exudative form in the fellow eye during the study period. No measurable evolution was found in consecutive fundus autofluorescence and indocyanine green angiography images. Total drusen area in CFP images presented no significant increase between each visit and the subsequent (p=0.100), but significant increase was found between the first and the last (p=0.048). IGC images: 23% of patients presented a normal exam; observations have shown that the hot spots or areas and the hypofluorescent spots or areas where either attributable to drusens or drusenoid pigmentary epitelium dettachments or did not correspond to any lesion visible in the CFP images; the sensitivity of the overall exam result was 88% and the specificity of presence of early hot spots or areas was 97%. FAF Abstract Multimodal Image: Assessment of Risk Factors for the Development of Choroidal Neovascularisation in the Fellow Eye images: the distribution of patterns for the cases that converted and those that did not is significantly different. CFP images: drusen area was significantly higher in the group of patients which did not convert when considering total area (p=0.012), sub-field 4 (p=0.020) and sub-field 5 (p=0.039); two main groups were identified by hierarchical cluster analysis, with Ward's linkage, when considering total area of drusens of the last visit available; principal component analysis identified total area as the most characterizing feature; no significant differences were found between the groups in what concerns the areas of drusen with specific sizes (C1: p=0.28; C2: p=0.11; C3: p=0.31); no correlation was found between age or total drusen area and conversion, and these two variables are not correlated. Conclusion The percentage of patients which converted during the study (34%) exceeds the expected rate for this time period. In what concerns evolution, further study including a longer follow-up period is suggested as it was only apparent for total drusen area calculated in CFP images between the first and last visits available. FAF images: FAF may be an interesting exam for predicting AMD progression but further studies are necessary to pinpoint the characterizing patterns. ICG images: the most characterizing features for conversion were the overall exam result and the presence of early hot spots or areas. CFP images: in this sample conversion does not occur for patients with the higher extension of lesions; no relation was found between conversion and patient’s age or drusen of specific sizes.<br>Estudar a progressão das alterações do fundo ocular através de imagem multimodal e identificar os fatores de risco para conversão em degenerescência macular relacionada com a idade (DMRI) neovascular no olho contra-lateral de doentes com DMRI exsudativa. Descrição da Amostra e Métodos Estudo de centro único, prospetivo, observacional, longitudinal com a duração de dois anos. Foram incluídos no estudo 62 doentes com o diagnóstico de DMRI neovascular num dos olhos e maculopatia relacionada com a idade no outro. Cada doente foi submetido a uma história médica e ocular detalhada, um exame oftalmológico completo com retinografia a cores (CFP), angiografia fluoresceínica (FA), angiografia com verde de indocianina (ICG) e imagem de autofluorescência do fundo na linha de base e repetidos com intervalos de seis meses. As CFP foram classificadas com recurso ao software RetmarkerAMD (Critical Software SA), um sistema de classificação assistida por computador baseado nas linhas guias do sistema de classificação internacional (Bird, Bressler et al., 1995). Resultados Cinquenta e três doentes completaram o período de seguimento de dois anos, dezoito dos quais converteram para a forma exsudativa no olho em estudo durante a duração do estudo. Não foi encontrada evolução mensurável em imagens consecutivas de angiografia com verde de indocianina e imagem de autofluorescência do fundo. A área total de drusens nas CFP não revelou aumento significativo entre visitas subsequentes (p=0,100), mas verificou-se a existência de aumento significativo de área entre a primeira e a última (p=0,048). Imagens de ICG: 23% dos casos apresentavam um exame normal; as zonas de hiperfluorescência e hipofluorescência foram atribuídas a drusens ou descolamentos drusenoides do epitélio pigmentar da retina ou não correspondiam a nenhuma lesão identificável nas imagens de CFP; a sensibilidade do resultado global do exame é de 88% e a Resumo Multimodal Image: Assessment of Risk Factors for the Development of Choroidal Neovascularisation in the Fellow Eye especificidade da presença de hiperfluorescências é de 97%. Imagens de FAF: foram encontradas diferenças significativas entre a distribuição de padrões de cada grupo (doentes que converteram e doentes que não converteram). Imagens de CFP: a área de drusens é significativamente maior no grupo de doentes que não converteu quando se considera a área total (p=0,012), sub-área 4 (p=0,020) e sub-área 5 (p=0,039); foram identificados dois grandes grupos através da análise hierárquica de agrupamentos, com ligação de Ward, quando se considera a área total de drusens da última visita disponível; a análise de componentes principais identificou a área total como a variável mais caracterizadora; não foram encontradas diferenças significativas entre os grupos no que diz respeito à área de drusens com tamanhos específicos (C1: p=0,28; C2: p=0,11; C3: p=0,31); não se encontrou correlação entre a idade ou a área total de drusens e a conversão e estas duas variáveis não estão correlacionadas entre si. Conclusão A percentagem de doentes que converteu durante o estudo (33%) é superior à esperada para este período de tempo. No que diz respeito à evolução, parece pertinente realizar estudos com período de seguimento mais alargado uma vez que esta só foi manifesta para a área total de drusens calculada através das imagens de CFP entre a primeira e a última visitas disponíveis. Imagens de FAF: a imagem de FAF pode ser interessante para prever a progressão da DMRI mas são necessários mais estudos para determinar os padrões caracterizadores. Imagens de ICG: os achados mais caracterizadores para a conversão são o resultado global do exame e a presença de hiperfluorescências precoces. Imagens de CFP: nesta amostra a conversão não ocorre nos doentes com extensão de lesões superior; não se encontrou relação entre a conversão e a idade ou drusens de tamanhos específicos

LA MESURE DU VOLUME SANGUIN CEREBRAL (VSC) PAR IMAGERIE PAR RESONANCE MAGNETIQUE (IRM) PERMET D'ETUDIER L'ANGIOGENESE TUMORALE. DANS CETTE THESE, UNE METHODE IRM, DITE METHODE T1 STATIONNAIRE RAPIDE (RSST1) POUR QUANTIFIER LE VSC EST PROPOSEE. LE PRINCIPE REPOSE SUR LES PROPRIETES DE LA RELAXATION LONGITUDINALE AVEC DES AGENTS DE CONTRASTE (AC) PARAMAGNETIQUES INTRAVASCULAIRES ET SUR UN MODELE DU CERVEAU BI-COMPARTIMENTAL EXTRA/INTRAVASCULAIRE SANS ECHANGE D'EAU. LA METHODE A ETE VALIDEE SUR DES RATS SAINS A 2.35T (VSC: 2 A 3%) ET LA SENSIBILITE EVALUEE SOUS HYPERCAPNIE (AUGMENTATION DU VSC DE 1%/MMHG CO2). POUR EVALUER L'EFFICACITE D'UN TRAITEMENT ANTITUMORAL, DES AC NE S'EXTRAVASANT PAS DURANT LA MESURE A TRAVERS UNE BARRIERE HEMATOENCEPHALIQUE (BHE) LESEE SONT NECESSAIRES. DEUX AC EXPERIMENTAUX, LE GD-ACX ET LE SINEREM, ONT ETE ETUDIES SUR DEUX MODELES DE RAT GLIOME C6 ET RG2. AVEC LE GD-ACX, LES MESURES ONT ETE CONFRONTEES A UNE ANALYSE MORPHOMETRIQUE DE LA MICROVASCULARISATION SUR DES COUPES IMMUNO-HISTOLOGIQUES. LES MESURES AVEC LE SINEREM ONT NECESSITE LE DEVELOPPEMENT D'ACQUISITIONS A TEMPS D'ECHO COURT ET ONT ETE COMPAREES A CEUX OBTENUS PAR LA METHODE DELTAR2* UTILISANT LE MEME AC. POUR DES AC QUI S'EXTRAVASENT (GD-DOTA ADMIS EN CLINIQUE), UTILISANT UNE ANALYSE PHARMACOCINETIQUE A DEUX COMPARTIMENTS, LES ACQUISITIONS DE LA METHODE RSST1 CONDUISENT A LA MESURE DU VSC ET AU COEFFICIENT DE TRANSFERT Κ LIE A LA PERMEABILITE DE LA BHE. EN CONCLUSION, LA METHODE RSST1, METHODE QUANTITATIVE DE MESURE DE VSC, PERMET AVEC DES AC APPROPRIES, DE REALISER DES ETUDES LONGITUDINALES DE L'ANGIOGENESE TUMORALE ET D'ACCEDER A LA PERMEABILITE VASCULAIRE.

"This work arose from anecdotal evidence for the existence of anti-angiogenic factors in shark cartilage powders. The project aimed to elucidate the mechanisms of anti-angiogenesis of shark cartilage, identify at least one novel anti-angiogenic factor from shark cartilage, and search for other natural anti-angiogenic factors in cartilage of other species." -- abstract.. This work arose from anecdotal evidence for the existence of anti-angiogenic factors in shark cartilage powders. The project aimed to elucidate the mechanisms of anti-angiogenesis of shark cartilage, identify at least one novel anti-angiogenic factor from shark cartilage, and search for other natural anti-angiogenic factors in cartilage of other species. The goals of the work have been successfully achieved. Firstly, a large number of commercially available shark cartilage powders have been investigated and it was found that 22% of samples tested showed no anti-angiogenic activity (CAM assay), while the remaining 78% showed variable anti-angiogenic activity. Secondly, cartilage from other species including emu, ostrich, deer, camel, crocodile and kangaroo have been investigate, and most were found to be bioactive in anti-angiogenesis. The screening potency of the bioactive agents not only varied between different cartilage depots in the one species but also varied between species. Bioactive fractions comparable in activity to those of shark were obtained from crocodile ischeum. In contrast, cartilage fractions obtained from the ostrich sternum and camel nasal septum did not show any anti-angiogenic activities. Mammalian representatives (deer, camel and kangaroo) in this study all had cartilage depots which provided variable bioactivity. Kangaroo rib and camel ear seem likely to be promising substitutes for shark cartilage if considering the efficiency of cartilage collection and availability and together with potency. Thirdly, mechanisms of anti-angiogenesis of shark cartilage have been investigated, it was found that shark cartilage inhibits angiogenesis on CAM by blocking heparin-binding of bFGF with heparin sulfate proteoglycans (HSPGs) on the surface of endothelial cells involving nitric oxide (NO). Finally, two active fractions have been isolated from shark cartilage, high anti-angiogenic activity has been shown in the bioassay, two proteins purified from these two active fractions were demonstrated on SDS-PAGE. The implications of these findings in terms of further scientific research and the cartilage trade are discussed_

La rétinopathie induite par l’oxygène (RIO) est un modèle animal semblable aux rétinopathies vue chez l’homme. Dans ce modèle, une destruction des microvaisseaux rétiniens est suivie d’une néovascularisation pathologique qui chez l’homme peut mener à un détachement de la rétine et subséquemment une perte de vision. Afin de remédier à cette revascularisation anarchique, un traitement de cellules souches (hématopoïétiques et mésenchymateuses) a été effectué chez des souris soumises à ce modèle. Les cellules injectées ont pu migrer à la rétine et induire une revascularisation saine (surtout les cellules souches mésenchymateuses). L’injection du milieu de culture de ces cellules induit aussi une revascularisation semblable à celle vue chez les souris traitées avec les cellules indiquant que l’effet thérapeutique des cellules semble être accompli par l’entremise de facteurs paracrines. Ces résultats suggèrent que ces cellules peuvent jouer un rôle au niveau de l’angiogénèse et indiquent un potentiel thérapeutique pour les rétinopathies.<br>Oxygen induced retinopathy (OIR) is an animal model that mimics the developing phases of retinopathies seen in humans such as diabetic retinopathy and retinopathy of prematurity. An initial destruction of retinal microvasculature is followed by pathological neovascularization that can lead to retinal detachment in humans and therefore blindness. Utilizing bone marrow derived stem cells (mesenchymal and hematopoietic), we aimed to repopulate the retina with normal vessels which are affected in the OIR model. Cells injected into the vitreous migrated to the retina and reduced both the area of vasoobliteration and neovascularization. Injection of conditioned cell medium also induced proper vascular repair similar to that seen in mice injected with cells indicating that the cells therapeutic effect is achieved through paracrine action. These results suggest that bone marrow stem cells play a role in angiogenesis and could be a potential therapeutic aid in treating retinopathies.

Bei angeborenen Herzfehlern, die bei 1 bis 1,2 % aller Lebendgeburten auftreten und so-mit die häufigste behandlungsbedürftige Organfehlbildung darstellen, kommen regelhaft kardiovaskuläre Implantate im Rahmen der chirurgischen bzw. interventionellen Therapie zum Einsatz. Hierzu zählen u. a. Shunts, Patches und Okkluder, die aus verschiedenen Implantatmaterialien hergestellt werden. Das Ziel dieser Arbeit bestand darin – basierend auf histologischen Untersuchungen – Unterschiede bzw. Gemeinsamkeiten bezüglich der Biokompatibilität nicht-metallischer Implantatmaterialien zu prüfen, da eine bewusste Materialauswahl kardiovaskulärer Implantate zur Therapie angeborener Herzfehler zu besseren Langzeitergebnissen der Implantate beitragen kann. Untersucht wurden Implantate, die im Rahmen von Korrekturoperationen entnommen wurden und anschließend im Forschungslabor für Pädiatrische Kardiologie und Intensivmedizin der Universitätsmedizin Göttingen ausgewertet wurden: Shunts aus PTFE (n = 21, durchschnittliche Implantationszeit: 18 Monate), Patches aus PTFE (n = 13, durchschnittliche Implantationszeit: 247 Monate) und Polyester (n = 4, durchschnittli¬che Implantationszeit: 321 Monate) sowie Okkluder aus PTFE (n = 3, durchschnittliche Implantationszeit: 74 Monate), Polyester (n = 9, durchschnittliche Implantationszeit: 30 Monate) und PVA (n = 2, durchschnittliche Implantationszeit: 23 Monate). Zur Herstellung histologischer Präparate wurden metallhaltige Implantate (Okkluder) sowie solche mit bereits makroskopisch sichtbarer Verkalkung in Methylmethacrylat-Kunstharz eingebettet und anschließend gesägt und geschliffen, sodass sie lichtmikroskopisch ausgewertet werden konnten. Die anderen Implantate wurden in Paraffin eingebettet und geschnitten. Neben konventionellen Färbungen zur Übersicht und Darstellung von Verkalkungen wurden immunhistochemische Färbungen eingesetzt. Unabhängig vom Implantatmaterial konnte regelhaft eine endothelialisierte und neovaskularisierte Pseudointima, hauptsächlich am ehesten aus Myofibroblasten und Fibroblasten bestehend, dargestellt werden. Das im Implantatmaterial neu gebildete Gewebe bestand hauptsächlich aus Fibroblasten und war neovaskularisiert. Implantatassoziierte, chronische Entzündungsreaktionen – getragen durch Makrophagen und Lymphozyten – sowie Fremdkörperreaktionen – getragen durch FKR – waren bei den Polyester- und PVA-Implantaten stärker ausgeprägt als bei den PTFE-Implantaten. Verkalkungen in Pseudointima- und Implantatgewebe wurden bei den Polyester-Implantaten ab einer Implantationszeit von 3 Jahren und 4 Monaten, bei den PTFE-Implantaten ab einer Implantationszeit von 5 Jahren und 10 Monaten beobachtet. Die durch Polyester hervorgerufene, stärker ausgeprägte Entzündungsreaktion ist als Ursache der zu einem früheren Zeitpunkt einsetzenden Verkalkung von Polyester-Implantaten anzusehen. Während bei den Polyester-Implantaten häufig eher ungleichmäßig verteilte und unregelmäßig geformte, punktförmige Verkalkungen bis hin zu kleinen Kalkaggregaten in Pseudointima- und Implantatgewebe vorhanden waren, wiesen die PTFE-Implantate zumeist gleichmäßige, großflächig-konfluierende Verkalkungen auf. Es konnte gezeigt werden, dass bei Implantaten, die Polyester- oder PTFE-Anteile enthal¬ten, mittelfristig mit der Entwicklung von lokalen Verkalkungen zu rechnen ist, die im Langzeitverlauf zu Komplikationen führen können. Dies muss bei der Implantatauswahl beachtet werden. Möglicherweise kann in Zukunft durch die Entwicklung neuartiger Materialien eine Verminderung der Verkalkungstendenz, zum Beispiel durch Biodegra¬dierbarkeit des Implantatmaterials, erreicht werden.