In dieser Dissertation wurden murine Aorta-Explantate im experimentellen Ansatz des "Aortic Ring Assay" über elf Tage kultiviert, erstmalig die Differenzierung zu F4/80(+)-Makrophagen gezeigt und eine nahezu vollständige Depletion dieser Zellen durch Clodronat-Liposomen bewirkt. Diese Adventitia-generierten Makrophagen wurden als die Hauptquelle des lokal gebildeten VEGF nachgewiesen. Die daraus resultierende Depletion des parakrin wirkenden VEGF resultierte in einer teilweisen Konservierung der CD34(+) „Vaskulogenen Zone“ der aortalen Adventitia. Zu der Bestätigung dieser Ergebnisse wurde experimentell über den VEGF-Rezeptor-Blocker E7080 in das VEGF-VEGFR-2 System eingegriffen. Die Versuchsansätze mit diesem Rezeptorblocker resultierten in einem ähnlichen Ergebnis wie die Versuche unter Makrophagen-Depletion. / In this dissertation murine aortic explants were cultivated for eleven days, using the method of “Aortic Ring Assay”. For the first time the differentiation to F4/80(+) macrophages and its depletion via clodronate liposomes could be described. These adventitia derived macrophages were identified to be the main source of the local VEGF. The depletion resulted in a lack of paracrine operating VEGF, thus partly preserving the CD34(+) vasculogenic zone within the aortal adventitia. To confirm these findings the VEGF-Receptor was blocked due to the VEGF-Receptor blocker E7080. These experiments resulted in almost the same findings compared to macrophage depletion.

Die Identifizierung endogener Stammzellen mit kardiogenem Potenzial und die Möglichkeit, deren Differenzierung zu steuern, würde einen Meilenstein in der kardioregenerativen Therapie darstellen. Innerhalb der Gefäßwand konnten unterschiedliche Stamm- und Vorläuferzellen identifiziert werden, die sog. Gefäßwand-residenten Stammzellen (VW-SCs). Zuletzt konnten aus CD34(+) VW-SCs, ohne genetische Manipulation, Kardiomyozyten generiert werden. Zusätzlich fungiert die Gefäßwand als Quelle inflammatorischer Zellen, die essenziell für die kardiogene Differenzierung der VW-SCs zu sein scheinen. Ziel dieser Arbeit war es, das Verhalten von CD44(+) VW-SCs zu untersuchen, um herauszufinden, inwieweit dieser Stammzelltyp eine endogene Generierung von Kardiomyozyten unterstützen könnte. Dabei wurde mit infarzierten Mäuseherzen, dem Aortenringassay (ARA) und dem kardialen Angiogeneseassay (CAA) gearbeitet. Sowohl in vivo in ischämischen Arealen infarzierter Mäuseherzen als auch ex vivo im CAA kam es zu einem signifikanten Anstieg von CD44(+) Zellen. Mittels Färbungen auf CD44 und Ki-67 konnte die Teilungsfähigkeit dieser Zellen demonstriert werden. Ex vivo ließen sich aus CD44(+) Zellen F4/80(+) Makrophagen generieren. Die CD44(+) VW-SCs können sich dabei sowohl zu pro-inflammatorischen iNOS(+) M1- als auch zu anti-inflammatorischen IL-10(+) M2-Makrophagen differenzieren. Eine Modulation der kardialen Inflammation könnte einen entscheidenden Einfluss auf die Kardiomyogenese haben. Unter VEGF-A kam es im CAA zu einer deutlichen Zunahme von CD44(+) Zellen. Unter Lenvatinib blieb das kardiale Sprouting gänzlich aus, die Anzahl der CD44(+) Zellen stagnierte und die VW-SCs verblieben in ihren physiologischen Nischen innerhalb der Gefäßwand. Warum es nach einem MI kaum zu einer funktionellen Herzmuskelregeneration kommt, ist weiterhin unklar. Die therapeutische Beeinflussung koronaradventitieller CD44(+) VW-SCs und inflammatorischer Prozesse könnte dabei zukünftig eine wichtige therapeutische Option darstellen. / The identification of endogenous stem cells with cardiogenic potential and the possibility to control their differentiation would represent a milestone in cardioregenerative therapy. Within the vascular wall, different stem and progenitor cells could be identified, the so-called vascular wall-resident stem cells (VW-SCs). Most recently, cardiomyocytes could be generated from CD34(+) VW-SCs, without genetic manipulation. In addition, the vascular wall acts as a source of inflammatory cells which appear to be essential for cardiogenic differentiation of VW-SCs. The objective of this work was to investigate the behavior of CD44(+) VW-SCs to see to what extent this stem cell type could support endogenous generation of cardiomyocytes. This was done using infarcted mouse hearts, the aortic ring assay (ARA), and the cardiac angiogenesis assay (CAA). There was a significant increase in CD44(+) cells in vivo in ischemic areas of infarcted mouse hearts and ex vivo in the CAA. A double staining for CD44 and Ki-67 demonstrated the ability of these cells to proliferate. Ex vivo, F4/80(+) macrophages could be generated from CD44(+) cells. Thereby, the CD44(+) VW-SCs can differentiate into both pro-inflammatory iNOS(+) M1 and anti-inflammatory IL-10(+) M2 macrophages. Modulation of cardiac inflammation may have a critical impact on cardiomyogenesis. Under VEGF-A, there was a clear increase in CD44(+) cells in the CAA. Under lenvatinib, cardiac sprouting was completely absent, the number of CD44(+) cells stagnated, and VW-SCs remained in their physiological niches within the vessel wall. Why there is little functional myocardial regeneration after MI remains unclear. Therapeutic manipulation of coronary adventitial CD44(+) VW-SCs and inflammatory processes may represent an important therapeutic option in the future.

Freie Universiẗat, Diss., 2005--Berlin. / Dateiformat: zip, Dateien im PDF-Format. Erscheinungsjahr an der Haupttitelstelle: 2005.

The perivascular adventitia (PA) senses and responds to injuries in blood vessels and the tissues they feed. Cells in the PA form the outermost vascular layer, joining the circulatory system to other organs. Housing hematopoietic, mesenchymal and neuronal cells allows flexible adventitial responses to diverse perturbations. However, the PA response can also be pathogenic. Thickening of the adventitia may drive ischemia and hypertension. It can also be a niche for local lymphocyte priming in diseases such as idiopathic pulmonary arterial hypertension. Despite their importance, PA contributions to skin diseases were understudied. The hypothesis that contrasting two cutaneous diseases, scleroderma and discoid lupus erythematosus (DLE), would illuminate discrete PA alterations was explored. Vascular changes are prominent, but distinct, in both diseases. Studying perivascular adventitial changes in these diseases may yield insights into both dermal and vascular pathologies. PA fibroblasts in healthy human skin were phenotypically distinct from the surrounding dermal fibroblasts. In both scleroderma and DLE, PA fibroblasts expanded and expressed surface markers not observed in healthy skin including vascular cell adhesion molecule 1 (VCAM1), podoplanin (PDPN) and the p75 low affinity nerve growth factor receptor (NGFR). Elaborated networks of PA fibroblasts in DLE expressed VCAM1 and enmeshed dense, T cell-rich infiltrates. Transcriptional analyses indicated positive correlations between VCAM1, T cell chemoattractants and interleukin (IL)-15, which promotes their survival. Activated PA fibroblasts in DLE likely create a supportive niche for T cells infiltrating the skin. In contrast, enlarged PA fibroblast networks in scleroderma expressed NGFR in the absence of leukocyte infiltrates. This PA fibroblast phenotype was shared among reparative and pathologic scarring, and four dermal tumors. NGFR is a mesenchymal stem cell (MSC) marker, and expanded NGFR+ mesenchymal cells were immediately adjacent to cluster of differentiation (CD)34+ and CD73+ PA MSC. Expression of NGFR by PA fibroblasts is likely associated with reparative responses. Different stimuli induced VCAM1 and NGFR on cultured human dermal fibroblasts, supporting these as discrete activation states. In conclusion, these studies demonstrated the responsive and plastic nature of human dermal PA mesenchymal cells, and pointed to connections with vascular alterations in skin diseases.

Congenital heart defects are the most common category of birth defects, mostly affecting the blood vessels, walls, or valves of the heart. For example, pulmonary atresia occurs when the connection between the right ventricle to the main pulmonary artery is not fully formed. A heart defect such as pulmonary atresia may need surgery to close up any malformations in walls and blood vessels, and unfortunately, because the patients are infants, they will need to undergo several surgeries in their lifetime to accommodate a heart patch that will fit the size of their hearts at each stage of their life. A better solution would be to create a biomimetic vascular patch that could be anatomically accepted by the patient’s body as its own, allowing it to grow with the patient without the residue of scar tissue. Instead of propagating scar tissue in the area, it would propagate healthy cells that would integrate into the surrounding tissue. For this to become a reality, one strategy for a biomimetic vascular patch would be to build it like a blood vessel in layers, beginning with the tunica adventitia. The goal of this thesis is to engineer and design the foundation for a biomimetic vascular patch with a crimped, collagen-integrated scaffold, focusing on optimizing the mechanical properties of the hybrid structure. The crimped structure, using sine waves generated from Python code and fabricated with bioprinting technology, mimics the natural formation of collagen fibers in native blood vessels. Additionally, testing the scaffolds on the Instron allows for characterization of the mechanical behaviors of an optimal and repeatable foundation for a tissue-engineered tunica adventitia. / 2023-01-22T00:00:00Z

No / Tunica adventitia, the outer layer of blood vessels, is an important structural feature, predominantly consisting of collagen fibrils. This study uses pseudostatic atomic force microscopy (AFM) nanoindentation at physiological conditions to show that the distribution of indentation modulus and viscous creep for the tunica adventitia of porcine aorta and pulmonary artery are distinct. Dynamic nanoindentation demonstrates that the viscous dissipation of the tunica adventitia of the aorta is greater than the pulmonary artery. We suggest that this mechanical property of the aortic adventitia is functionally advantageous due to the higher blood pressure within this vessel during the cardiac cycle. The effects on pulsatile deformation and dissipative energy losses are discussed.

Made available in DSpace on 2016-01-26T12:51:07Z (GMT). No. of bitstreams: 1 batigalia_tese_parte1.pdf: 948858 bytes, checksum: b48f5591911812d8f298686183d84a2a (MD5) Previous issue date: 2003-03-14 / Sociedade de Cardiologia do Estado de São Paulo / Introduction: About half of the cases of atherosclerotic coronary disease (a mean of 15% of women deaths and 25% of men) cannot be explained by most of the known risk factors. Coronary vasa vasorum are associated with coronary artery disease; however, their anatomy and physiopathology are not well clear. Objective: The aim of this study was to carry out a post mortem stereological study of adventitial vasa vasorum in different histopathological degrees of coronary atherosclerosis intending to correlate vasa vasorum, myocardial infarction physiopathology and histopathological degrees of atherosclerosis. Method: Ten consecutive autopsies of adults (5 men, 5 women, from 35 to 83 years-old, frozen at 4o C) were performed. Six proximal, medium and distal biopsies of the anterior and posterior interventricular coronary branches (at intervals of 1.5 cm) were performed per autopsy (a total of 60 coronary biopsy fragments). Fragments were processed by histological routine technique and cut in 4 fragments of 4 mm thickness. The first two consecutive histological fragments were stained by hematoxylin-eosin, and the two remaining by Masson´s trichrome. The fragments were histopathologically analysed according to Stary´s coronary atherosclerosis classification and examined by Zeiss Jenaâ, a light microscope with a bright chamber attached a Zeissâ micrometer scale, to outline adventitial vasa vasorum as well as to measure the coronary intraluminal diameter and the medial thickness. Intersection points of vasa vasorum with Merzâ´s grille were manually counted. For all types of vasa vasorum, points on Merzâ´s grille were counted to obtain the following stereological parameters of vasa vasorum: diameter, wall thickness, volumetric and superficial density, and adventitial connective tissue density. Parametric data were analysed by Pearson s linear correlation and principal component analysis. Agreement in determining coronary atherosclerosis degree in laminas stained by hematoxylin-eosin or Masson´s trichrome was assessed by kappa statistics. Differences among variables at each atherosclerosis degree was assessed by analysis of variance or Kruskal-Wallis test. Results: Coronary intraluminal diameter correlated negatively with coronary medial thickness and number of adventitial vasa vasorum (r>0.50; P-value<0.05). These correlations may be explained by sex, age and coronary atherosclerosis degree (r>0.50; P-value<0.05). All stereological parameters of vasa vasorum correlated negatively with coronary intraluminal diameter and positively with medial thickness, both explained by sex and atherosclerosis degree (r>0.50; P-value<0.05). The size of all types of vasa vasorum augmented proportionally to atherosclerosis histopathological degree aggravation. Kappa statistics for hematoxylin-eosin and Masson´s trichrome presented agreements varying from substantial or good to almost perfect or fine . All variables presented significant differences since the degree II of atherosclerosis. Conclusions: Coronary medial thickness and number of vasa vasorum correlated negatively with coronary intraluminal diameter. These correlations may be explained by sex and coronary atherosclerosis degree. Stereological parameters of vasa vasorum (except coronary adventitial connective tissue density) correlated positively with number of adventitial vasa vasorum as well as medial thickness, and negatively with coronary intraluminal diameter. Both correlations were determined by degree of atherosclerosis. Venular rupture in vulnerable atherosclerotic plaques may be associated with myocardial infarction arising since the degree II of atherosclerosis. / Introdução: Cerca de 50% dos casos de doença arterial coronária aterosclerótica (15% das mortes masculinas e 25% femininas) não são explicados pelos clássicos fatores de risco. Vasa vasorum coronários podem associar-se à aterosclerose coronariana; contudo, sua anatomia e fisiopatologia não estão completamente elucidadas. Objetivos: Realizar estudo estereológico dos vasa vasorum da túnica externa de artérias coronárias autopsiadas buscando correlação entre vasa vasorum, fisiopatologia do infarto do miocárdio e graus histopatológicos de aterosclerose. Material e Método: Em dez autópsias consecutivas de adultos (5 homens, 5 mulheres, 35 a 83 anos, congelados a 4º C) efetuaram-se biópsias proximal, média e distal dos ramos interventriculares anterior e posterior em cada autópsia, a cada 1,5 cm, totalizando 6 biópsias por autópsia. Cada fragmento foi processado histologicamente com cortes sucessivos de 4 cm de espessura, totalizando 4 fragmentos (24 fragmentos por autópsia). Os dois primeiros fragmentos foram corados em hematoxilina-eosina e os dois últimos em tricrômico de Masson. Lâminas histológicas foram diagnosticadas histopatologicamente quanto ao grau de aterosclerose coronariana pela classificação de Stary e examinadas em microscópio de luz Zeiss Jena com câmara clara e escala micrométrica Zeissâ para delineamento dos vasa vasorum da túnica externa coronária e mensuração do diâmetro do lúmen coronário e da espessura da túnica média coronária. Pontos dos delineamentos dos vasa vasorum sobre a grade estereológica foram manualmente contados obtendo-se diâmetro do lúmen, espessura da parede e densidades volumétrica e superficial dos vasa vasorum, e densidade do tecido conectivo da túnica externa. Dados paramétricos foram analisados por correlação linear de Pearson e análise de componentes principais, concordância no diagnóstico do grau aterosclerótico pela estatística kappa, e diferenças entre valores das variáveis a cada grau aterosclerótico por análise de variância ou teste de Kruskal-Wallis Resultados: Diâmetro do lúmen coronário correlacionou-se negativamente com espessura da túnica média e número de vasa vasorum da túnica externa (r>0,50; valor-p<0,05), com correlações explicadas pelo sexo e grau de aterosclerose (r>0,50; valor-p<0,05). Parâmetros estereológicos dos vasa vasorum correlacionaram-se negativamente com diâmetro do lúmen coronário e positivamente com espessura da túnica média, com correlações explicadas pelo grau de aterosclerose (r>0,50; valor-p<0,05). Tamanho de todos os tipos de vasa vasorum aumentou proporcionalmente ao agravamento das lesões ateroscleróticas. Estatística kappa para lâminas histológicas coradas em hematoxilina-eosina ou em tricrômico de Masson apresentou concordâncias variando de substancial ou boa a quase perfeita ou ótima . Todas as variáveis envolvidas apresentaram diferenças significativas a partir do grau II de aterosclerose coronariana. Conclusões: Espessura da túnica média e número de vasa vasorum da túnica externa correlacionaram-se negativamente com diâmetro do lúmen coronário, com correlações explicadas pelo sexo e grau de aterosclerose. Parâmetros estereológicos (exceto densidade do tecido conectivo da túnica externa) variaram proporcionalmente com espessura da túnica média e com número de vasa vasorum, e inversamente com diâmetro do lúmen coronário, com correlações explicadas pelo grau histopatológico de aterosclerose. Tamanho de cada tipo de vasa vasorum aumentou proporcionalmente à progressão dos graus ateroscleróticos. Ruptura venular precoce em placas ateroscleróticas vulneráveis poderia propiciar infarto do miocárdio desde o grau II ou III de aterosclerose.