Technological insights into a histopathological and protein composition analysis of aortic aneurysms in Marfan syndrome

[eng] The aorta is the main artery of the body, whose function is to collect the blood ejected by the heart and distribute it to all tissues. The elastic degree of the aortic wall necessary to withstand blood pressure is mainly provided by the distribution of collagen fibres and elastic lamellae. Fibrilin-1 is an essential protein of the elastic lamellae, which is mutated in Marfan syndrome (MFS), a connective tissue disorder characterized by the formation of aortic aneurysms with a high risk of mortality. Multiphoton microscopy and the microCT, among other approaches, have been used to evaluate the structural characteristics of the aortic tissue, the but there is currently little information about the three-dimensional histopathological structure of the MFS aorta. Besides, the differences in the protein content with respect to the healthy aorta have been analysed by means of studies that used conventional cell culture, neglecting the effect of the mechanical forces that the vascular smooth muscle cells (VSMC) constantly perceive in vivo due to blood pressure. In this context, we have developed four innovative technologies that have allowed us to generate new data on the histopathological structure and the protein content of the aorta MFS in comparison to healthy samples: - A multiphoton microscopy and image processing methodology was applied to MFS mouse aortae to visualise and analyse the microscale morphology of the elastic lamellae. Bigger and more abundant fenestrae were observed in the aorta of MFS compared to those of the WT. With this results, fenestrae become potential markers of lamellar damage in MFS. - A microCT and image processing protocol to evaluate the histological integrity of the aortic wall. This technology was applied to MFS and WT mice aortae, and an increase in the area and the thickness of the tunica media and other histological parameters was observed in relation to age progression (3, 6, and 9 months (mo)). This increase was parallel between WT and MFS, but the SMF 9mo showed values significantly higher than the WT 9m. Therefore, we hypothesise that the MFS aorta may undergo an accelerated middle-ageing process. - A bioreactor of mechanical stretching to examine the protein content of VSMCs of healthy human aorta and MFS cultivated under in vivo-like mechanical conditions. The MFS cells showed an altered reaction to the administration of stretch, not related to the content or distribution of phenotypic markers. Therefore, the MFS VSMCs show altered cell-matrix communication not linked to the phenotype. - Finally, by means of the systematic review of the scientific literature, the most complete human aortic tissue proteome to date was generated. It is divided into a healthy and an aneurysmal databases, containing 919 and 724 different proteins, respectively. The application of these technologies has provided new knowledge in the field of vascular biology, especially with regard to Marfan syndrome.[cat] L'aorta és la principal artèria del cos, la funció de la qual és recollir la sang expulsada pel cor i distribuir-la cap a tots els teixits. El grau elàstic de la paret aòrtica necessari per a suportar la pressió sanguínia ve donat principalment per la distribució de fibres de col·lagen i lamel·les elàstiques. La fibrilina-1 és una proteïna essencial de les lamel·les elàstiques, la qual es troba mutada en la síndrome de Marfan (SMF), un trastorn del teixit connectiu caracteritzat per la formació d’aneurismes d’aorta amb alt risc de mortalitat. Per a avaluar les característiques estructurals del teixit aòrtic s’han utilitzat, entre d’altres, la microscòpia multifotó i el microCT, però avui encara hi ha poca informació sobre l'estructura histopatològica tridimensional de l'aorta SMF. D'altra banda, s’han analitzat les diferències en el contingut proteic respecte l’aorta sana mitjançant estudis que empraven cultiu cel·lular convencional, descuidant l'efecte de les forces mecàniques que les cèl·lules musculars llises vasculars (VSMC) perceben constantment in vivo a causa de la pressió arterial. En aquest context, hem desenvolupat quatre tecnologies innovadores que han permès generar noves dades sobre l'estructura histopatològica i el contingut proteic de l'aorta SMF en comparació a mostres sanes: - Una metodologia de microscòpia multifotó i de processament d'imatges es va aplicar a aortes de ratolins SMF per a visualitzar y analitzar la morfologia microscala de les lamel·les elàstiques. Es van observar fenestres més grans i abundants en l'aorta de SMF en comparació amb les del tipus WT; esdevenint les fenestres potencials marcadors del dany lamel·lar en la SMF. - Un protocol de microCT i processament d'imatges per a avaluar la integritat histològica de la paret aòrtica. Aquesta tecnologia es va aplicar a aortes de ratolins SMF i WT, i es va observar un increment de l'àrea i el gruix de la túnica media i d’altres paràmetres histològics en relació amb la progressió en edat (3, 6 i 9 mesos (mo)). Aquest increment fou paral·lel entre WT i SMF, però el SMF 9mo mostrà valors significativament més alts que els WT 9mo. Per tant, plantegem la hipòtesi que l'aorta SMF deu patir un procés accelerat d'envelliment a edat adulta mitjana. - Un biorreactor d'estirament mecànic per a examinar el contingut proteic de VSMCs d’aorta humana sana i SMF cultivades sota un ambient mecànic similar al real. Les cèl·lules SMF van mostrar una reacció alterada a l'administració de l'estirament, no relacionada amb el contingut ni distribució de marcadors fenotípics. Per tant, les VSMCs de SMF mostren una comunicació cèl·lula-matriu alterada no associada al fenotip. - Finalment, mitjançant la revisió sistemàtica de la literatura científica es va generar el proteoma de teixit aòrtic humà més complet de l’actualitat, dividit entre les bases de dades sana i aneurismàtica, que contenen 919 i 724 proteïnes diferents, respectivament. L'aplicació d'aquestes tecnologies ha proporcionat nous coneixements al camp de la biologia vascular, especialment pel que fa a la síndrome de Marfan

Development of microfluidic devices with 3D collagen gels for traction force microscopy

In the past few decades, it has been widely demonstrated that cells constantly exert traction forces on the cells and matrix surrounding them. This behavior is fundamental in physiological and pathological processes such as embryogenesis and metastasis, therefore studying how cells interact with their environment is vitally important to understand these processes. To contribute to this research field, the present master project aims to adapt a given microfluidic device to the traction force microscopy technique, which is currently the most reliable approach for measuring cell forces. It is of great interest to quantify the forces exerted by cells seeded inside this microfluidic device because this specific device allows the deposition of collagen in a 3D arrangement, which resembles better the cell’s physiological environment than traditional 2D cell cultures. Device adaptation consisted in introduction of fluorescent microbeads into the collagen matrix, and subsequent confocal microscopy imaging of the cultured cells. Both stages required optimization of diverse features, for example: bead size and concentration, cell viability and labeling, fluorescence staining complications, etc. After numerous experiments and information search, the aforementioned features were improved, and ultimately, the microfluidic device was successfully adapted to traction force microscopy. Thus, the final assays produced useful data for performing the cell force calculation

High-Resolution morphological approach to analyse elastic laminae injuries of the ascending aorta in a murine model of Marfan Syndrome

In Marfan syndrome, the tunica media is disrupted, which leads to the formation of ascending aortic aneurysms. Marfan aortic samples are histologically characterized by the fragmentation of elastic laminae. However, conventional histological techniques using transverse sections provide limited information about the precise location, progression and 3D extension of the microstructural changes that occur in each lamina. We implemented a method using multiphoton excitation fluorescence microscopy and computational image processing, which provides high-resolution en-face images of segmented individual laminae from unstained whole aortic samples. We showed that internal elastic laminae and successive 2(nd) laminae are injured to a different extent in murine Marfan aortae; in particular, the density and size of fenestrae changed. Moreover, microstructural injuries were concentrated in the aortic proximal and convex anatomical regions. Other parameters such as the waviness and thickness of each lamina remained unaltered. In conclusion, the method reported here is a useful, unique tool for en-face laminae microstructure assessment that can obtain quantitative three-dimensional information about vascular tissue. The application of this method to murine Marfan aortae clearly shows that the microstructural damage in elastic laminae is not equal throughout the thickness of the tunica media and in the different anatomical regions of the ascending aorta

Cine y Biología: Una experiencia docente realizada por los estudiantes

El artículo narra una experiencia de un ciclo de cine organizada por los propios estudiantes susceptible de ser reconocida para obtener créditos de libre elección. La actividad fue realizada durante el curso 2009-10 en los estudios de Biología de la Universidad Pompeu Fabra (UPF) de Barcelona.La organización de la actividad y la evaluación de los alumnos matriculados fueron llevadas a cabo por los estudiantes organizadores bajo la supervisión del decano de la facultad.La experiencia fue evaluada positivamente por el alto número de matriculados, por la participación de los mismos y por el resultado de la evaluación de las memorias que tenían que realizar los matriculados. La actividad podría considerarse como un buen ejemplo de implicación de los propios estudiantes en su formación, tanto técnica como humana.</p

MicroCT imaging reveals differential 3D micro-scale remodelling of the murine aorta in ageing and Marfan syndrome

Aortic wall remodelling is a key feature of both ageing and genetic connective tissue diseases, which are associated with vasculopathies such as Marfan syndrome (MFS). Although the aorta is a 3D structure, little attention has been paid to volumetric assessment, primarily due to the limitations of conventional imaging techniques. Phase-contrast microCT is an emerging imaging technique, which is able to resolve the 3D micro-scale structure of large samples without the need for staining or sectioning. Methods: Here, we have used synchrotron-based phase-contrast microCT to image aortae of wild type (WT) and MFS Fbn1C1039G/+ mice aged 3, 6 and 9 months old (n=5). We have also developed a new computational approach to automatically measure key histological parameters. Results: This analysis revealed that WT mice undergo age-dependent aortic remodelling characterised by increases in ascending aorta diameter, tunica media thickness and cross-sectional area. The MFS aortic wall was subject to comparable remodelling, but the magnitudes of the changes were significantly exacerbated, particularly in 9 month-old MFS mice with ascending aorta wall dilations. Moreover, this morphological remodelling in MFS aorta included internal elastic lamina surface breaks that extended throughout the MFS ascending aorta and were already evident in animals who had not yet developed aneurysms. Conclusions: Our 3D microCT study of the sub-micron wall structure of whole, intact aorta reveals that histological remodelling of the tunica media in MFS could be viewed as an accelerated ageing process, and that phase-contrast microCT combined with computational image analysis allows the visualisation and quantification of 3D morphological remodelling in large volumes of unstained vascular tissues

High-Resolution Morphological Approach to Analyse Elastic Laminae Injuries of the Ascending Aorta in a Murine Model of Marfan Syndrome

In Marfan syndrome, the tunica media is disrupted, which leads to the formation of ascending aortic aneurysms. Marfan aortic samples are histologically characterized by the fragmentation of elastic laminae. However, conventional histological techniques using transverse sections provide limited information about the precise location, progression and 3D extension of the microstructural changes that occur in each lamina. We implemented a method using multiphoton excitation fluorescence microscopy and computational image processing, which provides high-resolution en-face images of segmented individual laminae from unstained whole aortic samples. We showed that internal elastic laminae and successive 2nd laminae are injured to a different extent in murine Marfan aortae; in particular, the density and size of fenestrae changed. Moreover, microstructural injuries were concentrated in the aortic proximal and convex anatomical regions. Other parameters such as the waviness and thickness of each lamina remained unaltered. In conclusion, the method reported here is a useful, unique tool for en-face laminae microstructure assessment that can obtain quantitative threedimensional information about vascular tissue. The application of this method to murine Marfan aortae clearly shows that the microstructural damage in elastic laminae is not equal throughout the thickness of the tunica media and in the different anatomical regions of the ascending aorta.Peer Reviewe

Cinema and Biology: A teaching experience carried out by students

[ES] El artículo narra una experiencia de un ciclo de cine organizada por los propios estudiantes susceptible de ser reconocida para obtener créditos de libre elección. La actividad fue realizada durante el curso 2009-10 en los estudios de Biología de la Universidad Pompeu Fabra (UPF) de Barcelona. La organización de la actividad y la evaluación de los alumnos matriculados fueron llevadas a cabo por los estudiantes organizadores bajo la supervisión del decano de la facultad. La experiencia fue evaluada positivamente por el alto número de matriculados, por la participación de los mismos y por el resultado de la evaluación de las memorias que tenían que realizar los matriculados. La actividad podría considerarse como un buen ejemplo de implicación de los propios estudiantes en su formación, tanto técnica como humana. [EN] The paper recounts the experience of a cycle of film showings organized by the students themselves, which was also recognized as being an activity whereby elective credits could be obtained. The activity was carried out during the 2009-10 academic year in the Biology degree at the Pompeu Fabra University (UPF) in Barcelona. The organization of the activity and the assessment of the students enrolled were undertaken by the Student Organizers under the supervision of the Dean of the Faculty of Health and Life Sciences. The experience was deemed to be highly successful for the following reasons: the number of students enrolled on it, the notable participation and the result of the assessment of the reports handed in by the students. The activity could be considered a good example of students’ involvement in their own training, both technical and human

MicroCT imaging reveals differential 3D micro-scale remodelling of the murine aorta in ageing and Marfan syndrome

Aortic wall remodelling is a key feature of both ageing and genetic connective tissue diseases, which are associated with vasculopathies such as Marfan syndrome (MFS). Although the aorta is a 3D structure, little attention has been paid to volumetric assessment, primarily due to the limitations of conventional imaging techniques. Phase-contrast microCT is an emerging imaging technique, which is able to resolve the 3D micro-scale structure of large samples without the need for staining or sectioning.J.L.-G. is supported by a Spanish Government predoctoral fellowship FPI (BES-2013-065962 and EEBB-I-16-11080), and by a Boehringer Ingelheim Fonds travel grant for her stay at Diamond synchrotron. The Manchester X-Ray Imaging Facility is funded in part by the Engineering and Physical Sciences Research Council [ESPRC] (grant numbers EP/F007906/1, EP/F001452/1 and EP/I02249X/1). This study was supported by grants SAF2015-64136-R and SAF2017-83039-R to G.E., and partially supported by grants TIN2014-52923-R, Maria de Maeztu Units of Excellence Programme/MDM-2015-0502, and FEDER to B.B. The Diamond Manchester collaboration awarded synchrotron beam time to the authors (session ID MT12199-1). C.D. was supported by an EPSRC & MRC Centre for Doctoral Training (CDT) Regenerative Medicine (EP/L014904/1) studentship. P.W. is grateful for a European Research Council grant (Correl-CT grant No 695638). G.E. thanks Damelys Alejandra Fumero for her casual economic contribution to his research