Obstructive Sleep Apnea (OSA) is one of the most frequent chronic disease affecting nearly one billion people worldwide. OSA corresponds to the repetitive occurrence of complete (apneas) and incomplete (hypopneas) pharyngeal collapses during sleep leading to cycling hypoxia-re-oxygenation sequences called intermittent hypoxia. OSA is a growing health concern, highlighted by a societal and economic burden close to hypertension or stroke. OSA is associated and modulate severity of various chronic diseases including metabolic syndrome, nonalcoholic fatty liver diseases and cardiovascular disease. The landmark feature of OSA is a chronic intermittent hypoxia (CIH) associated with apneas/hypopneas leading to activation of the hypoxia-inducible transcription factors (HIF) directly responsible for multiple organ damages including liver disease. The interconnections between hypoxia and circadian rhythms has become a hot research topic in physiological science. This link is largely explained by transcriptional and epigenetic mechanisms. However, data are limited regarding these interactions in OSA, a particularly relevant disease since it induces intermittent hypoxia during a specific circadian time period (sleep). This is therefore an important emerging area to explore and the main goal of this project is to demonstrate that reprogramming of circadian homeostasis by CIH is a key mechanism underlying OSA-related organ injury with a specific focus on liver disease. Given the intimate link between hypoxia, circadian rhythms and chromatin dynamics, by understanding how these components act as a coordinated network TEMPORISE may provide novel insight into how these factors contribute to liver disease in general and more specifically in OSA. First, we aim to explore the kinetics of liver disease evolution under intermittent hypoxia exposure and to characterize the timing of circadian rhythm perturbation by CIH. Second, we wish to understand molecular mechanisms underlying circadian clock dysregulation at the onset of liver disease, through a molecular study of the interaction between the circadian clock and HIF-1 and the associated transcriptional and epigenetics consequences. Third, we want to analyze the effect of circadian clock and HIF-1 disruption on CIH-induced liver disease development through histological characterization and the use of appropriate knockout mouse models. The combined expertise of the coordinator and the partners are major assets for the TEMPORISE project. This project will take advantage of state-of-the art technics such as epigenomics, transcriptomics, proteomics and bioinformatics approaches. Altogether, we expect that unraveling novel mechanisms interconnecting CIH and circadian clock defects will provide new insight in the understanding of liver diseases development and pave new avenues towards pharmacological interventions.

Obstructive sleep apnea is a growing worldwide health problem. The landmark feature of OSA is a chronic intermittent hypoxia (CIH) responsible for multiple organ damages including heart diseases. Under several pathophysiological conditions such as aging, vWAT senescence drives myocardial alterations through the release of profibrotic factors. Our lab has demonstrated that CIH profoundly alters both vWAT and heart structure and function, but little is known regarding their interactions in the context of CIH. Thus, our primary objective is to demonstrate that CIH induces a premature vWAT senescent phenotype, responsible for subsequent heart dysfunction. From this, we aim at i) bringing the proof-of-concept that strategies targeting CIH-induced vWAT senescence exert beneficial effects on cardiac alterations, and ii) identifying CIH-specific circulating biomarkers that may ultimately contribute to cardiac dysfunction.

Scientific rationale and hypothesis: Obstructive sleep apnea (OSA) is a worldwide public health problem, affecting at least 5% of the general population and characterized by repetitive upper airway occlusions during sleep leading to intermittent hypoxia (IH). Patients with OSA exhibit increased cardiovascular morbidity and mortality, including systemic hypertension, coronary heart disease, arrhythmias and stroke. Treatment of patients with continuous positive airway pressure (CPAP) improves quality of life and daytime sleepiness. However, the major barriers to CPAP treatment are adherence and its failure to alter metabolic or inflammatory markers in obese OSA. Thus a combination of multiple modalities of treatment is needed. The nonmuscle myosin light chain kinase (nmMLCK) isoform is a protein that contributes to endothelial cell-cell junction opening, monocyte migration and therefore participates in inflammation. We have described cardiovascular dysfunction, mainly endothelial dysfunction, associated with inflammation and oxidative stress in OSA patients. Also, circulating levels of microparticles expressing CD62L and derived from activated leukocytes positively correlates with oxyhemoglobin desaturation index (ODI) whereas endothelial nitric oxide production correlated negatively with both CD62L+ microparticles and ODI. IH in mice induces early hemodynamic alterations and cardiovascular remodeling and systemic inflammation associated with overexpression of NF-kappaB, ICAM-1 and RANTES/CCL5 as well as T lymphocyte infiltration. Interestingly, we reported that although deletion of nmMLCK does not affect physiological cardiovascular parameters in vivo, it protects against endotoxic shock through the decrease of the up-regulation of NF-kappaB expression and activity, inducible nitric oxide synthase expression, and level of oxidative stress in the vascular media. nmMLCK deficiency or inhibition has been reported to attenuate systemic, lung and atherosclerotic inflammation. From these observations, one can advance the hypothesis that nmMLCK might be an attractive target to fight against the occurrence of inflammation and the vascular outcomes of OSA. Objectives and aims: By combining basic research in animal models, molecular studies in different types of cell culture and biological and genetic analyses in humans, our aims are: (i) to validate the molecular implication of nmMLCK in IH-associated inflammation and vascular effects, (ii) to evaluate nmMLCK as a biological marker of the severity of OSA, and (iii) to analyze the association between nmMLCK polymorphism and OSA. The goal is to translate the knowledge on nmMLCK to the bedside in order to improve the management and treatment of patients of OSA.

The passive stiffness of elastic arteries is mainly determined by two major extracellular matrix proteins of the arterial wall, i.e. elastin and collagen. Elastin provides reversible extensibility during cyclic loading of the cardiac cycle, while collagen provides stiffness and strength at high pressures. Loss of elasticity and induced consequences on the vascular function are observed in normal ageing, and in syndromic elastogenesis-related genetic diseases which include Williams-Beuren syndrome, supra-valvular aortic stenosis and autosomal dominant cutis laxa. Recent studies have also shown it can be associated with pathological conditions such as the sleep apnea syndrome (SAS). Knowing that SAS concerns about 10% of the general population and affects 20% in the elderly, in 2015 in France, 837,000 people were treated for SAS. Williams-Beuren syndrome is a rare disease but represents 3,000 people in France and is estimated at 300,000 patients over the world. The age-related cardiovascular dysfunctions, involving elastic fibre alterations, concern a large and growing part of the population. If we could introduce new elastin in the existing elastic scaffold of arteries, and if this provides increased elasticity, we would have a revolutionary treatment related to a very large market in the pharmaceutical field. Arterylastic project aims at restoring the function and mechanical properties of blood vessels using an original synthetic elastic protein recently developed by the principal investigator (“DHERMIC”, ANR 2012-2016). This represents a very important breakthrough since this compound might serve as an elastic molecular prosthesis in tissues to compensate or restore the lack of elasticity. To reach this ambitious goal, the Arterylastic project will investigate and optimize the following properties of this synthetic compound: - delivering the right signal to cells with no deleterious effect; - reaching the right location through the endothelial barrier; - being integrated into elastic fibres within vascular walls; - improving arterial wall elasticity and/or physiological parameters in relevant animal models; - developing an injectable formulation of the compound with a pharmaceutical grade. We have large convincing data set available on skin for the synthetic elastic protein (DHERMIC project) and we recently obtained preliminary very promising results for blood vessel integration in fish and mouse. The success of the proposal will also rely on strong complementarities between the three internationally recognised academic partners: LBTI is expert in biology of elastic fibres and therapeutic engineering; HP2 has a strong expertise in vascular physiology and in elastin hemizygous and intermittent hypoxia mice models mimicking sleep apnea syndrome (SAS); and ARMINES-CIS is a very active laboratory in the field of biomechanics and numerical modelling of soft tissues. To ensure a certified pharmaceutical grade formulation, the FRI Pharm facility core from Lyon hospital is involved in the project. After achieving the scientific investigations, a partnership with a pharmaceutic company will be addressed to allow for further industrial and clinical developments. Moreover, a startup company will be created in order to accelerate the industrial transfer and at mid-term to participate in local socio-economic dynamism.

Single domain antibodies (sdAb or nanobodies) based imaging agent directed at the inflammatory marker Vascular Cell Adhesion Molecule 1 (VCAM-1) have recently been validated by the LRB (partner 1) for the non-invasive nuclear imaging of atherosclerosis and liver inflammation in mice. The lead compound (cAbVCAM1-5) has been produced according to good manufacturing practice and a phase I/II clinical trial is underway at Grenoble-Alpes University Hospital. The aim of the present project is to further evaluate its capabilities using preclinical mice models in order to pave the way of future clinical evaluations. To fulfill this objective, we will evaluate its ability to detect and quantify chronic inflammation in various pathological settings and organs. Moreover, we will evaluate the potency of anti-VCAM-1 sdAb for magnetic resonance imaging (MRI), as an alternative to nuclear imaging, using biodegradable microparticles developed by partner 4 (PhIND). cAbVCAM1-5 will therefore be evaluated in a mouse model of spondyloarthritis in collaboration with the T-Raig team (Lab TIMC, partner 2), in mice models of myocardial infarction with HP2 laboratory (partner 3) and in mice models of neuroinflammation in collaboration with PhIND. Upon completion of this research project, the consortium will have investigated the potential of the anti-VCAM-1 sdAb in 3 pathological settings that could potentially benefit from improved diagnostic and prognostic imaging biomarkers for the management of patients. The results will therefore allow determining relevant strategies for the evaluation of this novel imaging agent, in particular for the design of phase II clinical trials. Moreover, it will provide a better understanding of the tissue and subcellular distribution of this imaging agent, and will explore the possibility to extend its field of application using MRI as a second imaging modality.