Propriétés mécaniques des Cellules Musculaires Lisses isolées issues d’aortes humaines saines et anévrysmales

Smooth muscle cells (SMC) play a major role in the mechanobiological response of the aortic wall. Certain influencing factors, such as pathological conditions, lead to deregulation of the mechanosensitivity and mechanotransduction processes of the SMC. These changes are reflected in particular by a phenotypic change of the SMC, from contractile to synthetic, in order to repair the tissue which is perceived as deficient. Remodeling induces stiffening and weakening of the wall. Given the lack of quantitative data about the mechanical properties of human aortic SMCs, we undertook their characterization in normal and pathological conditions, in order to better anticipate alterations leading to injury. Accordingly, this thesis presents a set experimental studies carried out on SMCs from primary cultures of human origin, from healthy and aneurysmal donors. The characterization of SMCs is based on two engineering techniques: traction force microscopy to measure the basal tone, and nanoindentation by atomic force microscopy to measure the apparent stiffness of isolated SMCs. Results show increased tensile forces in aneurysmal cells, but no significant change in stiffness. Future work aims to characterize the mechanical properties in the cytoskeleton of these cells and to assess how mechanical stimuli affect these properties.La cellule musculaire lisse (CML) joue un rôle majeur dans la réponse biomécanique de la paroi aortique. Certains facteurs d’influence, telles que des conditions pathologiques, entraînent une dérégulation des processus de mécanosensibilité et de mécanotransduction de la CML. Ces changements se traduisent notamment par un changement de phénotype des CML contractiles vers synthétiques, afin de réparer le tissu qu’elles perçoivent comme défectueux. Le remodelage induit une rigidification et une fragilisation de la paroi qui devient alors plus sujette au risque de rupture. Toutefois, au vu du manque de données quantitatives à propos des propriétés mécaniques des CML aortiques humaines, leur caractérisation nous est apparue essentielle en conditions normales et pathologiques, afin de mieux anticiper les altérations conduisant à une lésion. Dans cette perspective, cette thèse présente un ensemble de travaux expérimentaux menés sur des CML issues de culture primaire d’origine humaine, issues de donneurs sains et anévrismaux. La caractérisation des CML repose sur deux techniques d’ingénierie : la traction force microscopie pour mesurer le tonus basal, et la nanoindentation par microscopie à force atomique pour accéder à la rigidité apparente de CML isolées. Les résultats montrent une augmentation des forces de traction dans les cellules anévrysmales, mais pas de changement de rigidité significatif. Les perspectives envisagées sont de caractériser les propriétés mécaniques dans le cytosquelette de ces cellules et d’évaluer comment les stimulations mécaniques affectent ces propriétés

Mechanical properties of Smooth Muscle Cells isolated from healthy and aneurysmal human aortae

La cellule musculaire lisse (CML) joue un rôle majeur dans la réponse biomécanique de la paroi aortique. Certains facteurs d’influence, telles que des conditions pathologiques, entraînent une dérégulation des processus de mécanosensibilité et de mécanotransduction de la CML. Ces changements se traduisent notamment par un changement de phénotype des CML contractiles vers synthétiques, afin de réparer le tissu qu’elles perçoivent comme défectueux. Le remodelage induit une rigidification et une fragilisation de la paroi qui devient alors plus sujette au risque de rupture. Toutefois, au vu du manque de données quantitatives à propos des propriétés mécaniques des CML aortiques humaines, leur caractérisation nous est apparue essentielle en conditions normales et pathologiques, afin de mieux anticiper les altérations conduisant à une lésion. Dans cette perspective, cette thèse présente un ensemble de travaux expérimentaux menés sur des CML issues de culture primaire d’origine humaine, issues de donneurs sains et anévrismaux. La caractérisation des CML repose sur deux techniques d’ingénierie : la traction force microscopie pour mesurer le tonus basal, et la nanoindentation par microscopie à force atomique pour accéder à la rigidité apparente de CML isolées. Les résultats montrent une augmentation des forces de traction dans les cellules anévrysmales, mais pas de changement de rigidité significatif. Les perspectives envisagées sont de caractériser les propriétés mécaniques dans le cytosquelette de ces cellules et d’évaluer comment les stimulations mécaniques affectent ces propriétés.Smooth muscle cells (SMC) play a major role in the mechanobiological response of the aortic wall. Certain influencing factors, such as pathological conditions, lead to deregulation of the mechanosensitivity and mechanotransduction processes of the SMC. These changes are reflected in particular by a phenotypic change of the SMC, from contractile to synthetic, in order to repair the tissue which is perceived as deficient. Remodeling induces stiffening and weakening of the wall. Given the lack of quantitative data about the mechanical properties of human aortic SMCs, we undertook their characterization in normal and pathological conditions, in order to better anticipate alterations leading to injury. Accordingly, this thesis presents a set experimental studies carried out on SMCs from primary cultures of human origin, from healthy and aneurysmal donors. The characterization of SMCs is based on two engineering techniques: traction force microscopy to measure the basal tone, and nanoindentation by atomic force microscopy to measure the apparent stiffness of isolated SMCs. Results show increased tensile forces in aneurysmal cells, but no significant change in stiffness. Future work aims to characterize the mechanical properties in the cytoskeleton of these cells and to assess how mechanical stimuli affect these properties

Traction Force Measurements of Human Aortic Smooth Muscle Cells Reveal a Motor-Clutch Behavior

International audienceThe contractile behavior of smooth muscle cells (SMCs) in the aorta is an important determinant of growth, remodeling, and homeostasis. However, quantitative values of SMC basal tone have never been characterized precisely on individual SMCs. Therefore, to address this lack, we developed an in vitro technique based on Traction Force Microscopy (TFM). Aortic SMCs from a human lineage at low passages (4-7) were cultured 2 days in conditions promoting the development of their contractile apparatus and seeded on hydrogels of varying elastic modulus (1, 4, 12 and 25 kPa) with embedded fluorescent microspheres. After complete adhesion, SMCs were artificially detached from the gel by trypsin treatment. The microbeads movement was tracked and the deformation fields were processed with a mechanical model, assuming linear elasticity, isotropic material, plane strain, to extract the traction forces formerly applied by individual SMCs on the gel. Two major interesting and original observations about SMC traction forces were deduced from the obtained results: 1. they are variable but driven by cell dynamics and show an exponential distribution, with 40% to 80% of traction forces in the range 0-10 µN. 2. They depend on the substrate stiffness: the fraction of adhesion forces below 10 µN tend to decrease when the substrate stiffness increases, whereas the fraction of higher adhesion forces increases. As these two aspects of cell adhesion (variability and stiffness dependence) and the distribution of their traction forces can be predicted by the probabilistic motor-clutch model, we conclude that this model could be applied to SMCs. Further studies will consider stimulated contractility and primary culture of cells extracted from aneurysmal human aortic tissue

Review of the Essential Roles of SMCs in ATAA Biomechanics

International audienceAortic Aneurysms are among the most critical cardiovascular diseases. The present study is focused on Ascending Thoracic Aortic Aneurysms (ATAA). The main causes of ATAA are commonly cardiac malformations like bicuspid aor-tic valve or genetic mutations. Research studies dedicated to ATAA tend more and more to invoke multifactorial eects. In the current review, we show that all these eects converge towards a single paradigm relying upon the crucial biome-chanical role played by smooth muscle cells (SMCs) in controlling the distribution of mechanical stresses across the aortic wall. The chapter is organized as follows. In section 6.2, we introduce the basics of arterial wall biomechanics and how the stresses are distributed across its dierent layers and among the main structural constituents: collagen, elastin, and SMCs. In section 6.3, we introduce the biome-chanical active role of SMCs and its main regulators. We show how SMCs actively regulate the distribution of stresses across the aortic wall and among the main structural constituents. In section 6.4, we review studies showing that SMCs tend to have a preferred homeostatic tension. We show that mechanosensing can be understood as a reaction to homeostasis unbalance of SMC tension. Through the use of layer-specic multiscale modeling of the arterial wall, it is revealed that the quantication of SMC homeostatic tension is crucial to predict numerically the initiation and development of ATAA

In Vitro Biological Effects of E-Cigarette on the Cardiovascular System—Pro-Inflammatory Response Enhanced by the Presence of the Cinnamon Flavor

The potential cardiovascular effects of e-cigarettes remain largely unidentified and poorly understood. E-liquids contain numerous chemical compounds and can induce exposure to potentially toxic ingredients (e.g., nicotine, flavorings, etc.). Moreover, the heating process can also lead to the formation of new thermal decomposition compounds that may be also hazardous. Clinical as well as in vitro and in vivo studies on e-cigarette toxicity have reported potential cardiovascular damages; however, results remain conflicting. The aim of this study was to assess, in vitro, the toxicity of e-liquids and e-cigarette aerosols on human aortic smooth muscle cells. To that purpose, cells were exposed either to e-liquids or to aerosol condensates obtained using an e-cigarette device at different power levels (8 W or 25 W) to assess the impact of the presence of: (i) nicotine, (ii) cinnamon flavor, and (iii) thermal degradation products. We observed that while no cytotoxicity and no ROS production was induced, a pro-inflammatory response was reported. In particular, the production of IL-8 was significantly enhanced at a high power level of the e-cigarette device and in the presence of the cinnamon flavor (confirming the suspected toxic effect of this additive). Further investigations are required, but this study contributes to shedding light on the biological effects of vaping on the cardiovascular system

In Vitro Biological Effects of E-Cigarette on the Cardiovascular System—Pro-Inflammatory Response Enhanced by the Presence of the Cinnamon Flavor

International audienceThe potential cardiovascular effects of e-cigarettes remain largely unidentified and poorly understood. E-liquids contain numerous chemical compounds and can induce exposure to potentially toxic ingredients (e.g., nicotine, flavorings, etc.). Moreover, the heating process can also lead to the formation of new thermal decomposition compounds that may be also hazardous. Clinical as well as in vitro and in vivo studies on e-cigarette toxicity have reported potential cardiovascular damages; however, results remain conflicting. The aim of this study was to assess, in vitro, the toxicity of e-liquids and e-cigarette aerosols on human aortic smooth muscle cells. To that purpose, cells were exposed either to e-liquids or to aerosol condensates obtained using an e-cigarette device at different power levels (8 W or 25 W) to assess the impact of the presence of: (i) nicotine, (ii) cinnamon flavor, and (iii) thermal degradation products. We observed that while no cytotoxicity and no ROS production was induced, a pro-inflammatory response was reported. In particular, the production of IL-8 was significantly enhanced at a high power level of the e-cigarette device and in the presence of the cinnamon flavor (confirming the suspected toxic effect of this additive). Further investigations are required, but this study contributes to shedding light on the biological effects of vaping on the cardiovascular system

Atomic Force Microscopy Stiffness Mapping in Human Aortic Smooth Muscle Cells

International audienceAortic smooth muscle cells (SMCs) play a vital role in maintaining mechanical homeostasis in the aorta. We recently found that SMCs of aneurysmal aortas apply larger traction forces than SMCs of healthy aortas. This result was explained by the significant increase of hypertrophic SMCs abundance in aneurysms. In this study, we investigate whether the cytoskeleton stiffness of SMCs may also be altered in aneurysmal aortas. For that, we use atomic force microscopy (AFM) nano-indentation with a specific mode that allows subcellular-resolution mapping of the local stiffness across a specified region of interest of the cell. Aortic SMCs from a commercial human lineage (AoSMCs, Lonza) and primary aneurysmal SMCs (AnevSMCs) are cultured in conditions promoting the development of their contractile apparatus, and seeded on hydrogels with stiffness properties of 12 kPa and 25 kPa. Results show that all SMCs exhibit globally a lognormal stiffness distribution, with medians in the range 10–30 kPa. The mean of stiffness distributions is 16 kPa in aneurysmal SMCs and 12 kPa in healthy cells, but the differences are not statistically significant due to the large dispersion of AFM nano-indentation stiffness. We conclude that the possible alterations previously found in aneurysmal SMCs do not affect significantly the AFM nano-indentation stiffness of their cytoskeleton

Characterization of micro/nano-rheology properties of soft and biological matter combined with a virtual reality haptic exploration

International audienceScience education is often limited by the complexity of making acute knowledge accessible and easily remembered by a larger public. We proposed another way to introduce and teach complex scientific subjects like cell biology and polymer micro-rheology using haptic display and virtual reality. We combined the advantages of the Atomic Force Microscopy (AFM) and Fluorescence Microscopy (FM) in order to get complementary real experimental data on an isolated animal adhering on a protein micro-pattern. We obtained high resolution mapping images of the cell morphology, architecture, and local mechanical properties. Then this set of data was implemented in a free simulation engine connected to a low-cost haptic device to create a virtual and interactive cell or polymer environment in order to provide a novel sensory approach of the cell biology and/or micro(bio)mechanics

Regulation of SMC traction forces in human aortic thoracic aneurysms

International audienc