Mechanosensitive ATP release in the lungs

L’ATP est bien connue pour son rôle de transporteur d'énergie à l’intérieur des cellules, mais en dehors de la cellule, elle agit en tant que molécule de signalisation extracellulaire. En se liant aux récepteurs purinergiques, l’ATP extracellulaire amorce la signalisation purinergique afin de réguler certains processus physiologiques et pathophysiologiques. Dans les poumons, l’ATP stimule la sécrétion de surfactant et promeut la clairance mucociliaire. Compte tenu du rôle critique de l’ATP extracellulaire dans les poumons, il est important de comprendre le mécanisme du relargage d’ATP cellulaire — la première étape de la signalisation purinergique. Parce que les forces mécaniques constituent le déclencheur principal du relargage d’ATP, cette thèse a pour but d’investiguer le(s) mécanisme(s) physiologique(s) et les sources cellulaires d’un tel relargage d’ATP mécanosensible. Cet ouvrage est divisé en trois parties : 1) Pour étudier les caractéristiques spatiales et temporelles du relargage d’ATP, j’ai développé une technique d’imagerie hautement sensible basée sur la bioluminescence de la luciférine-luciférase couplée avec un système de lentilles à grand champ de vision (WFOV, wide field of view) optimisant l’apport de lumière. Pour évaluer notre approche d’imagerie, j’ai soumis des cellules A549, dérivées d’un adénocarcinome pulmonaire humain, à un étirement ou un choc hypotonique de 50% pour déclencher un relargage d’ATP. J’ai démontré que notre technique nous permet de quantifier précisément la quantité et le taux (ou l’efflux) d’ATP s’échappant des cellules. Le WFOV constitue un outil essentiel utilisé dans les études décrites dans cette thèse pour déterminer le mécanisme et la source cellulaire du relargage d’ATP dans l’alvéole. 2) Afin d’examiner le mécanisme physiologique du relargage d’ATP induit par l’étirement dans les cellulaires alvéolaires primaires, j’ai déterminé les contributions individuelles des cellules alvéolaires de type 1 (AT1) en comparaison des cellules alvéolaires de type 2 (AT2). Pour ce faire, des cellules AT2 fraîchement isolées de poumons de rats ont été ensemencées sur une chambre flexible en silicone et cultivées jusqu’à sept jours, ce qui permettait aux cellules AT2 de se transdifférencier progressivement en cellules semblables aux cellules AT1. Le ratio des cellules alvéolaires (AT2:AT1), étant de 4:1 au jour 3, est devenu 1:4 au jour 7. La quantité d'ATP libérée diminuait avec le nombre décroissant de cellules AT2, les impliquant en tant que principale source pour le relargage d’ATP en réponse à un étirement. Alors que les modulateurs pharmacologiques des canaux d’ATP, carbenoxolone et probénécide, ne diminuaient pas la quantité d’ATP libérée, le BAPTA, un chélateur de calcium intracellulaire ([Ca2+]i), l’a significativement réduite. De même, ces trois modulateurs exercent des effets similaires sur les réponses calciques intracellulaires mesurées par le Fura-2, suggérant une connexion entre le relargage d’ATP et les niveaux de [Ca2+]i. 3) Pour explorer le rôle qu’ont les propriétés viscoélastiques de la membrane dans le relargage d’ATP mécanosensible, j’ai démontré qu’une déformation de 30% induisait un relargage d’ATP transitoire qui était accompagné d’une absorption d’iodure de propidium (PI, propidium iodide) chez des cellules AT2. Ceci est cohérent avec une rupture membranaire transitoire induite par une déformation, assez large pour le passage d’ATP et de PI. L’efflux d’ATP augmente aussi selon le taux de déformation, et la durée de déformation prolonge la demi-vie du relargage d’ATP. Donc, ces résultats fournissent des indices sur la manière dont l’étirement de la membrane viscoélastique peut mener au relargage d’ATP par un mécanisme alternatif impliquant une mécanoporation de la membrane cellulaire. Dans l’ensemble, ces résultats démontrent que le relargage d’ATP ne se produit pas à travers les canaux conduisant l’ATP mais plutôt par une mécanoporation transitoire de la membrane. D’autres études sur les dommages membranaires sont nécessaires pour mieux comprendre sa contribution dans le relargage d’ATP mécanosensible et les signaux de [Ca2+]i. De telles études élucideront la signalisation purinergique dans les organes qui sont constamment exposés à des contraintes physiques. Ceci pourrait suggérer des cibles/approches thérapeutiques pour moduler les impacts négatifs d’un relargage d’ATP excessif observés lors de certaines conditions pathologiques, telles que les lésions pulmonaires induites par la ventilation mécanique.ATP is widely known to be an energy carrier within cells, but outside of the cell, it acts as an extracellular signaling molecule. Upon binding to purinergic receptors, extracellular ATP initiates the purinergic signaling to regulate certain physiological and pathophysiological processes. In the lungs, ATP stimulates surfactant secretion and promotes mucociliary clearance. Given the critical role of extracellular ATP in the lungs, it is important to understand the mechanism of cellular ATP release — the first step of purinergic signaling. Because mechanical forces constitute the primary trigger of ATP release, this thesis aims to investigate the physiological mechanism(s) and cellular sources of such mechanosensitive ATP release. This work is divided into three parts: 1) To study the spatial and temporal characteristics of ATP release, I developed a highly sensitive imaging technique based on luciferin-luciferase bioluminescence coupled with a custom-designed lens system, which combined a wide field of view (WFOV) and high light-gathering power. To evaluate our imaging approach, I subjected A549 cells, derived from human lung adenocarcinoma, to stretch or 50% hypotonic shock to trigger ATP release. I demonstrated that our technique allows us to precisely quantify the amount and the rate (or efflux) of ATP escaping from cells. The WFOV constitutes an essential tool used in the studies described in this thesis to determine the mechanism and cellular source of ATP release in the alveolus. 2) To examine the physiological mechanism of stretch-induced ATP release in primary alveolar cells, I determined the individual contributions of alveolar type 1 (AT1) in comparison with alveolar type 2 (AT2) cells. To this end, freshly isolated AT2 cells from rat lungs were seeded on a flexible silicone chamber and were cultured for up to seven days, which allowed AT2 cells to progressively transdifferentiate into AT1-like cells. The ratio of alveolar cells (AT2:AT1), being 4:1 on day 3, became 1:4 on day 7. The quantity of released ATP decreased with the decreasing numbers of AT2 cells, implicating them as the main source of ATP release in response to stretch. While pharmacological ATP channel modulators, carbenoxolone and probenecid, did not diminish the amount of ATP release, BAPTA, an intracellular calcium ([Ca2+]i) chelator, significantly reduced it. Likewise, these three modulators had similar effects on intracellular calcium responses measured by Fura-2, suggesting a connection between ATP release and [Ca2+]i levels. 3) To explore the role of membrane viscoelastic properties in mechanosensitive ATP release, I demonstrated that a 30% strain induced transient ATP release that was accompanied by uptake of propidium iodide (PI) in AT2 cells. This is consistent with a strain-induced transient membrane rupture, big enough for the passage of ATP and PI. ATP efflux also increases with strain rate, and hold time prolongs the half-life of ATP release. Thus, these results provide clues on how stretching of the viscoelastic membrane may lead to ATP release via an alternate mechanism involving transient mechanoporation of the cell membrane. Overall, these findings demonstrate that stretch-induced ATP release does not occur through ATP-conducting channels but rather a transient membrane mechanoporation. Further studies on membrane injury induced by strain are needed to better understand its contribution to mechanosensitive ATP release and [Ca2+]i signaling. Such studies will elucidate purinergic signaling in organs that are constantly exposed to physical stresses. This could suggest novel therapeutic targets/approach to modulate the negative impacts of excessive ATP release observed under certain pathological conditions, such as ventilator-induced lung injury

Endoscopic, Anatomic OCT for Imaging and Compliance Measurement of Upper and Central Airways

Both acute airway injuries such as inhalation injury and prevalent but underdiagnosed diseases such as obstructive sleep apnea (OSA) lead not only to impaired quality of life but also to disability or even death. However, current techniques such as bronchoscopy, computed tomography and magnetic resonance imaging all have limitations, such as being semi-quantitative or the exposure to ionizing radiation or long scan times, when it comes to airway imaging. A modality that provides high-resolution, real-time, safe and minimally invasive imaging of the airways would be very beneficial in the diagnosis and treatment of airway diseases. Additionally, changes in the biomechanical properties of airway tissues associated with underlying pathophysiologic status of tissues have not been much explored. Thus, an imaging modality that also has the ability to perform elastography could be valuable in the diagnosis and treatment of inhalation injuries. Optical coherence tomography (OCT) is a rapidly developing imaging modality providing iv high-resolution and non-invasive imaging of tissue microstructure. To image the upper and central airways of pediatric patients, a specific type of OCT -- the swept-source anatomic optical coherence tomography (SSaOCT), which has a micron-level resolution and an imaging range over 10 mm is utilized. It allows direct visualization of features on airway walls as well as sub-surface structures such as cartilage and trachealis muscle. Moreover, aOCT together with a pressure catheter can be used to perform anatomic optical coherence elastography (aOCE) and measure airway compliance to predict the regions of the airway wall that are vulnerable to collapse. This provides additional diagnostic information of airways that is not easily achievable with other imaging modalities. In this dissertation, the design and performance of the two custom-built aOCT systems are described, and their ability to accurately measure airway geometry and compliance is investigated. Imaging of phantoms and animal specimens is performed, aOCE-derived compliance is calculated and the relationship between the compliance measurements and the severity of steam injury is evaluated. Results indicate that aOCT can perform accurate airway imaging as well as assess the compliance of airway tissues. The measured compliance of the airway could potentially be used as an index for grading and assessing the severity of injuries and thus aid in the diagnosis and treatment of airway inhalation injury.Doctor of Philosoph

Alveolar Microfluidic Systems for Study of Barrier Function, Cell Damage, and Migration at the Air-Blood Barrier.

The exchange of oxygen and carbon dioxide occurs across the air-blood barrier (or alveolar-capillary barrier). This barrier must be sufficiently thin to allow the passive diffusion, yet sufficiently strong to maintain a dry alveolar environment. When solid and fluid mechanical stresses damage the air-blood barrier’s integrity, edema fills this normally air-filled alveolar environment and pathology results. The specific mechanisms by which these stresses impact the cells of the air-blood barrier remain poorly understood. The role of solid mechanical stress (cyclic stretch) has been explored through traditional, culture techniques, but only recently have microfluidic systems allowed systematic exploration on combined solid and fluid stresses. Although such systems can be tailored to the biological phenomena being studied, key design parameters include: (i) two-layered channel design (to mimic “alveolar” and “endothelial” compartments), (ii) ability to convey combined solid and fluid stresses, (iii) co-culture, and (iv) the integration of biological sensors to detect real-time changes. A microfluidic “Alveoli-on-a-Chip” system was designed and fabricated. By varying the degree of fluid-filling within the “alveolar” channel, differential strain conditions were applied to alveolar epithelial cells. Experiments using this system, demonstrated significant increases in cell death and detachment in alveolar cell populations exposed to fluid and solid mechanical stresses compared to populations exposed solely to solid mechanical stresses. Because nearly all pathological processes of alveoli alter barrier permeability, detection of changes to the integrity of this barrier is an essential feature in alveolar models. A technique for embedding Ag/AgCl recording electrodes within a two-layered PDMS microsystem, allowing impedance to be measured across a porous cell culture membrane was also developed. This fabrication technique eliminated the need for direct deposition of recording electrodes onto the elastomer, avoiding the frequent and deep cracking pattern resulting from the modulus mismatch between conductive metals and PDMS polymer. The impact of mechanical stresses on the alveolar immune response was also studied by patterning alveolar macrophages onto confluent monolayers of alveolar epithelial cells using aqueous two-phase (ATPS) printing. Using this technique, increased migration rates in co-cultures experiencing physiologic stretch levels were demonstrated compared to migration in static cultures.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/97972/1/ndouvill_1.pd

Advancements and Breakthroughs in Ultrasound Imaging

Ultrasonic imaging is a powerful diagnostic tool available to medical practitioners, engineers and researchers today. Due to the relative safety, and the non-invasive nature, ultrasonic imaging has become one of the most rapidly advancing technologies. These rapid advances are directly related to the parallel advancements in electronics, computing, and transducer technology together with sophisticated signal processing techniques. This book focuses on state of the art developments in ultrasonic imaging applications and underlying technologies presented by leading practitioners and researchers from many parts of the world

A Microscale High-Throughput Phenotypic Assay to Evaluate ECM Remodeling in Pulmonary Fibrosis

In fibrotic disease, dysregulation of matrix remodeling generates excessive deposition of fibrous extracellular protein that can interfere with the architecture and function of tissue. Due to the substantial variety of factors that combinatorially influence extracellular matrix (ECM) turnover, there exists the need for a phenotypic assay to evaluate cumulative effects involving cell-mediated fibrinolysis and collagen deposition. The goal of this project was to develop a novel in vitro assay that mimics fibroblast-mediated remodeling of the provisional fibrin matrix, in order to establish a model system for fibrotic scar formation and evaluate potential therapies. This work introduces and evaluates new methods to analyze ECM turnover in a high- throughput, label-free format. An aqueous two-phase printing technique was established to enable generation of microscale fibroblast-laden fibrin gels, which resemble the provisional fibrin matrix in wound healing. In a first variant of the assay, addition of exogenous plasminogen enabled cell-mediated activation of plasmin for gradual degradation of the fibrin matrix. A second variation of the assay evaluated remodeling of the fibrin matrix through concurrent fibrinolysis and collagen deposition. Live-cell imaging provided time-course brightfield micrographs that were analyzed through automated image processing protocols for high- throughput evaluation of different stages of remodeling. By investigating the cumulative effects of fibrinolysis and collagen deposition on fibroblastic remodeling of fibrin, this assay may provide a new resource for advancing understanding of fibrosis pathogenesis and for evaluating potential anti-fibrosis therapeutics.Ph.D

Book of Abstracts 15th International Symposium on Computer Methods in Biomechanics and Biomedical Engineering and 3rd Conference on Imaging and Visualization

In this edition, the two events will run together as a single conference, highlighting the strong connection with the Taylor & Francis journals: Computer Methods in Biomechanics and Biomedical Engineering (John Middleton and Christopher Jacobs, Eds.) and Computer Methods in Biomechanics and Biomedical Engineering: Imaging and Visualization (JoãoManuel R.S. Tavares, Ed.). The conference has become a major international meeting on computational biomechanics, imaging andvisualization. In this edition, the main program includes 212 presentations. In addition, sixteen renowned researchers will give plenary keynotes, addressing current challenges in computational biomechanics and biomedical imaging. In Lisbon, for the first time, a session dedicated to award the winner of the Best Paper in CMBBE Journal will take place. We believe that CMBBE2018 will have a strong impact on the development of computational biomechanics and biomedical imaging and visualization, identifying emerging areas of research and promoting the collaboration and networking between participants. This impact is evidenced through the well-known research groups, commercial companies and scientific organizations, who continue to support and sponsor the CMBBE meeting series. In fact, the conference is enriched with five workshops on specific scientific topics and commercial software.info:eu-repo/semantics/draf