Translational and Mechanistic Study about Beta-1-Adrenergic Receptor Modulation on Neutrophils as a Therapy against Ischemia/Reperfusion Injury

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de Lectura: 24-02-2023This work received funding from the Instituto de Salud Carlos III (ISCIII; PI16/02110 and PT20/00044), the European Regional Development Fund (ERDF) “A way of making Europe", the Comunidad de Madrid (S2017/BMD-3867 RENIM-CM) cofunded with European structural and investment funds and by Agencia Estatal de Investigación (PID2019‐110369RB‐I00). The CNIC is supported by the ISCIII, the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (CEX2020-001041-S

Circulating microvesicles as mediators of acute pulmonary vascular inflammation

Acute lung injury (ALI) resulting from remote ‘indirect’ causes is a major problem in sepsis and systemic inflammatory response syndrome (SIRS) but the underlying mechanisms are poorly understood. Circulating microvesicles (MVs) have been implicated as long-range mediators of vascular inflammation and their role as biomarkers in sepsis and SIRS has been widely investigated in clinical studies in recent years. However, the in vivo functional roles of MVs in sepsis and ALI have received less attention. Specifically, the role of in vivo MVs in the development of sepsis/SIRS-induced indirect ALI has not been previously evaluated. We hypothesised that circulating microvesicles (MVs) play a crucial role in propagating inflammation to the lungs, contributing to the development of pulmonary vascular inflammation in indirect ALI. The overall aims of this project were to: 1) evaluate MV uptake by pulmonary vascular cells and the mechanisms involved, 2) characterise the intravascular production of MVs in animal models of sepsis and sterile extrapulmonary organ injury, and 3) identify the contribution of in vivo-derived circulating MVs to the development of indirect ALI. The major findings of this work were that during sub-clinical endotoxaemia in mice, lung-marginated Ly6Chigh monocytes become a major target for circulating MV uptake via a phosphatidylserine receptor mechanism1. In mouse models of sepsis and extrapulmonary organ injury, neutrophil- and monocyte-derived MVs were the predominant MV subtypes being produced during endotoxaemia, while platelet- and endothelial-derived MVs were predominant during kidney ischaemia reperfusion injury. When MVs obtained from plasmas of endotoxaemic mice were adoptively transferred to isolated perfused lungs (IPLs), they induced significant increases in lung oedema. Depletion of intravascular lung monocytes by treatment with clodronate liposomes resulted in the reversal of the oedema, demonstrating the role of monocytes in MV-induced ALI. To investigate the contribution of different circulating MV subtypes, we immunoaffinity isolated myeloid (CD11b+) and platelet (CD41+) MVs from endotoxaemic mouse plasmas and transferred these to the IPL. We found that myeloid-MVs induced significant lung oedema and potent release of soluble mediators, whereas platelet-MVs produced a statistically significant, but much lower level of oedema and negligible release of soluble mediators. In summary, these findings indicate an important role of myeloid-derived MVs, particularly those derived from neutrophils and/or monocytes, and their interaction with lung-marginated monocytes in the pathogenesis of pulmonary vascular inflammation in indirect ALI. Further work to elucidate the specific MV molecular effectors mechanism involved will facilitate an enhanced understanding of ALI pathobiology.Open Acces

Doctor of Philosophy

dissertationCapsaicin is the pungent compound in chili peppers. Capsaicin causes dosedependent respiratory and cardiovascular failure by all routes. The capsaicin receptor, TRPV1, is a ligand-gated calcium channel. TRPV1 is expressed in sensory neurons and various non-neuronal cells in the lung, including epithelial cells of the conducting airways and alveoli. In human lung bronchial epithelial and alveolar cells, plasma membrane and endoplasmic reticulum (ER) populations of TRPV1 differentially influence cytokine gene expression and cell death via changes in cytosolic calcium concentrations, but a precise mechanism for TRPV1-mediated cytotoxicity was previously undefined. This project investigated how prototypical and endogenous TRPV1 agonists damage lung cells. Structure activity relationships between cell death and capsaicinoids with varied potency, and the role of TRPV1 in lung injury due to unfettered systemic inflammation were studied. In vitro assays for cell viability and calcium flux, quantitative analysis of gene expression patterns, and mutagenesis of regulatory gene products demonstrated that TRPV1 activation caused extensive ER calcium efflux, ER stress, and cell death. A series of capsaicinoid analogues were developed to determine the specificity of TRPV1 activation and ER stress as a common mechanism of toxicity in various human lung cell types. Structural modifications to the vanilloid ring drastically reduced the ability of capsaicinoids to activate TRPV1 and, accordingly, both ER stress and cytotoxicity were attenuated. Molecular modeling of iv analogue-TRPV1 binding corroborated these results and highlighted key structural features of capsaicin required for TRPV1 activation and cytotoxicity. Endogenous TRPV1 agonists have recently been implicated as pneumotoxicants during systemic inflammation. Treatment of mice i.p. with lipopolysaccharides (LPS) promoted systemic inflammation and lung injury. TRPV1 knockout mice and mice co-treated with the TRPV1 antagonist LJO-328 were protected from lung injury and evidence of an ER stress response was diminished. The endovanilloid anandamide induced ER stress and lung cell death in vitro, but these effects were not blocked by TRPV1 antagonist cotreatment, suggesting that other endovanilloids may cause ER stress and lung injury in mice. The results of this project provide a concerted mechanism of TRPV1-mediated lung epithelial cell death and illustrate a potential role of TRPV1 and endovanilloids in lung injury

Dynamic Modeling and System Identification of the Human Respiratory System

The lungs are the primary organ of the respiratory system. Their main function is to provide freshly breathed oxygen (O²) to the blood capillaries, while taking carbon dioxide (CO²) from them and expelling it to the atmosphere. Lung conditions such as Acute Respiratory Distress Syndrome (ARDS), Idiopathic Pulmonary Fibrosis (IPF), Coronavirus Disease (COVID-19), etc., cause impaired gas exchange that is life-threatening. In this dissertation, I developed 1) a physiology-based dynamic pulmonary system to study the lung normo- and patho-physiology, and 2) a model-based constrained optimization algorithm to do parameter estimation in order to non-invasively assess lung health. The goals of this work are 1) to accomplish a respiratory personalized medicine example for clinical decision support, and 2) to further the understanding of respiratory physiology, via a mechanistic physiology-based model and system identification techniques. The mechanistic model presented in this thesis comprises six subsystems: 1) a lung mechanics module that computes airflow transport from the mouth and nose to the alveoli (gas exchange units), 2) a respiratory muscles and rib cage mechanics module that simulates the effect of the respiratory muscle contraction on the lungs and the rib cage, 3) a microvascular exchange system that describes fluid (water) and mass (albumin and globulin) transport between the pulmonary capillaries and the alveolar space, 4) an alveolar elasticity module that computes alveolar compliance as a function of the pulmonary surfactant concentration and the elastic properties of the lung tissue fiber, 5) a pulmonary blood circulation that describes blood transport from the heart to the pulmonary system, and 6) a gas exchange system that describes O² and CO² transport between blood in the pulmonary capillaries and gas in the alveoli. Each subsystem was developed based on the latest knowledge of lung physiology and was validated using patient data when available or published and validated physiology-based models. To our knowledge, the combined six-module model would be the most rigorous and expansive lung dynamic model in the literature. This dynamic respiratory system can be used to describe human breathing under healthy and diseased conditions. The model can readily be used to test different what-if scenarios to find the optimal therapy for the patients. Further, I tailor the proposed lung model and adopt system identification techniques for noninvasive assessment of the lung mechanical properties (resistance and compliance) and the patient breathing effort. Pulmonary syndromes or diseases, such as ARDS and COPD (Chronic Obstructive Pulmonary Disease) evoke alterations in lung resistance and compliance. These two parameters reflect, by and large, the state of health and functionality of the respiratory system. Hence tracking these two parameters can lead to better disease diagnosis and easier monitoring of the respiratory disease progression. For spontaneously breathing patients on ventilatory support, the estimation of the lung parameters is challenging due to the added patient’s breathing effort. This dissertation presents a model-based nonlinear constrained optimization algorithm to estimate, breath-by-breath, the lung resistance, the lung compliance, as well as the patient breathing effort due to the respiratory muscle activity, using readily available non-invasive measurements (airway opening pressure and airflow)

Acute lung injury in paediatric intensive care: course and outcome

Introduction: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) carry a high morbidity and mortality (10-90%). ALI is characterised by non-cardiogenic pulmonary oedema and refractory hypoxaemia of multifactorial aetiology [1]. There is limited data about outcome particularly in children. Methods This retrospective cohort study of 85 randomly selected patients with respiratory failure recruited from a prospectively collected database represents 7.1% of 1187 admissions. They include those treated with High Frequency Oscillation Ventilation (HFOV). The patients were admitted between 1 November 1998 and 31 October 2000. Results: Of the 85, 49 developed acute lung injury and 47 had ARDS. There were 26 males and 23 females with a median age and weight of 7.7 months (range 1 day-12.8 years) and 8 kg (range 0.8-40 kg). There were 7 deaths giving a crude mortality of 14.3%, all of which fulfilled the Consensus I [1] criteria for ARDS. Pulmonary occlusion pressures were not routinely measured. The A-a gradient and PaO2/FiO2 ratio (median + [95% CI]) were 37.46 [31.82-43.1] kPa and 19.12 [15.26-22.98] kPa respectively. The non-survivors had a significantly lower PaO2/FiO2 ratio (13 [6.07-19.93] kPa) compared to survivors (23.85 [19.57-28.13] kPa) (P = 0.03) and had a higher A-a gradient (51.05 [35.68-66.42] kPa) compared to survivors (36.07 [30.2-41.94]) kPa though not significant (P = 0.06). Twenty-nine patients (59.2%) were oscillated (Sensormedics 3100A) including all 7 non-survivors. There was no difference in ventilation requirements for CMV prior to oscillation. Seventeen of the 49 (34.7%) were treated with Nitric Oxide including 5 out of 7 non-survivors (71.4%). The median (95% CI) number of failed organs was 3 (1.96-4.04) for non-survivors compared to 1 (0.62-1.62) for survivors (P = 0.03). There were 27 patients with isolated respiratory failure all of whom survived. Six (85.7%) of the non-survivors also required cardiovascular support.Conclusion: A crude mortality of 14.3% compares favourably to published data. The A-a gradient and PaO2/FiO2 ratio may be of help in morbidity scoring in paediatric ARDS. Use of Nitric Oxide and HFOV is associated with increased mortality, which probably relates to the severity of disease. Multiple organ failure particularly respiratory and cardiac disease is associated with increased mortality. ARDS with isolated respiratory failure carries a good prognosis in children

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