Abstract:
Bone regeneration remains a significant clinical challenge, requiring a deeper understanding of the cellular and molecular mechanisms underlying bone repair. Osteocytes, which represent 90%-95% of bone cells, form a connective dendritic network and play a key role in skeletal homeostasis. Embedded within the mineralized matrix, they experience unique metabolic constraints, including reduced oxygen availability. Hypoxia may, therefore, influence their function, but its impact and potential role in regulating angiogenesis during bone regeneration remain poorly understood. This study aimed to investigate (1) how hypoxia affects osteocytes and (2) their paracrine communication with endothelial cells (ECs). To model this interaction, we cultured the osteocytic cell line murine long bone osteocyte-Y4 (MLO-Y4) under normoxia (21% O₂), moderate hypoxia (5% O₂), or severe hypoxia (1% O₂) and collected the conditioned media (CM) after 48 h. Mouse ECs (1 × 10(4) cells/cm(2)) were cultured for 48 h in a 1:1 ratio of EC medium and CM from normoxic or hypoxic (5% and 1% O₂) osteocytes. Following optimization of co-culture conditions, RNA sequencing was performed to evaluate osteocyte and ECs' gene expression changes. Our findings indicate that hypoxia contributes to the development of the osteocyte dendritic network and upregulates neurogenesis-associated genes. Moreover, oxygen levels differentially influence osteocyte behavior by modulating their hypoxic response and metabolism. Indeed, severe hypoxia (1% O₂) induces hypoxia-responsive genes and glycolysis more strongly than moderate hypoxia (5% O₂), resulting in distinct metabolic and transcriptional profiles. However, osteocyte-derived factors did not significantly alter gene expression in ECs, suggesting that paracrine signaling, which is possibly not modulated by hypoxia, is not the principal mode of communication involved. Further studies are required to clarify the mechanisms by which oxygen levels shape osteocyte physiology.

Abstract:
Pharmacokinetic factors play a critical role in cocaine use. Previous work mainly examined externally driven pharmacokinetic manipulations, showing that conditions producing rapid fluctuations in brain cocaine levels, such as intermittent versus continuous access, promote burst-like intake, increased motivation for cocaine, heightened relapse vulnerability, and persistent drug-seeking behavior. Importantly, even under identical access conditions, substantial inter-individual variability exists in both the amount and speed of cocaine self-administration. In contrast to access-related pharmacokinetic influences, the contribution of intrinsic pharmacokinetic factors remains largely unexplored. To address this gap, male rats self-administered cocaine (0.8 mg/kg/injection; maximum 35 injections/session) for up to 26 sessions. In a final session, all animals received eight non-contingent intravenous cocaine injections at 5-min intervals before brain collection. Cocaine and its metabolites (ecgonine methyl ester, benzoylecgonine, norcocaine) were quantified in eleven sampled brain regions using liquid chromatography-tandem mass spectrometry detection and related to previous self-administration behavior. Mean brain cocaine concentration matched predictions from a widely used pharmacokinetic model, yet marked inter-individual variability was observed. Lower cocaine levels and higher metabolite-to-cocaine ratios were associated with faster self-administration patterns, but not with the total amount of cocaine previously consumed. This suggests that the observed inter-individual pharmacokinetic variability is unlikely to result from differential metabolic adaptations driven by intake and may contribute to differences in cocaine intake patterns. Overall, inter-individual variability in cocaine pharmacokinetics in the rat exist and are related to differences in cocaine intake patterns, with rapid intake representing a known risk factor for problematic cocaine use in both rodents and humans.

Abstract:
Osteocytes embedded in mineralized bone have their network disrupted when a bone injury occurs. However, their role in regeneration is still unclear. Bone substitutes, including bioceramics and bone-derived extracts, are commonly implanted in critical-sized defects due to their bioactive properties that promote healing, yet few studies have examined their effects on osteocytes in vivo. We studied early repair phases of a critical defect model in adult male mice calvaria, with or without biomaterial implantation (beta-TCP, bovine bone). Using microCT we determined that, after 14 days, bone formation had mainly occurred along the surface of existing bone, increasing its thickness by a factor of 1.6, independently of the biomaterial presence. Using HE staining and fluorescence imaging, we described the newly formed bone and showed the presence of recently embedded osteocytes. We specifically collected osteocytes close, and distant from the defect, using laser-assisted microdissection and analyzed their gene expression. We show that IL6 was mainly dependent on the delay after surgery whereas Dmp1 was spatially regulated. Thus, even with limited bone formation in the defect, bone apposition occurs on the inner and outer periosteal surfaces of the calvaria, a phenomenon that may have been overlooked in the development of bone repair strategies.

Abstract:
Extracellular vesicles (EVs) are key mediators of maternal-foetal communication, regulating placental function and foetal development through the transfer of bioactive molecules. Although placental EVs play a crucial role in placental function during pregnancy, their contribution to foetal development, notably foetal brain, remains poorly understood. Human cytomegalovirus (HCMV) is the most common virus transmitted in utero and a leading cause of infectious brain malformations. Although certain central nervous system lesions caused by HCMV are explained, the neuropathogenesis of congenital infection remains poorly understood. In this study, we demonstrate that EVs from healthy placentas promote neurogenesis. However, EVs from HCMV-infected placentas lose this neurogenic potential, impairing differentiation and migration of neural stem cells, perturbations that may contribute to the neurodevelopmental defects observed in congenital HCMV infections. miRNA profiling revealed profound infection-induced changes, including the incorporation of viral miRNAs and dysregulation of host miRNAs involved in neurogenesis. These findings highlight the critical role of placental EVs in foetal brain development and their contribution to HCMV neuropathogenesis.

Abstract:
PURPOSE: We present here the technical feasibility of percutaneously retrieving cardiomyocytes (CMs) through the lumen of irrigated ablation catheters, with the aim of obtaining DNA of sufficient quality/quantity for allowing DNA amplification, screening, and derived genetic analysis. METHODS: Irrigated conventional catheters for ablation were used for creating endocardial right ventricular voltage maps in 38 patients with suspected or proved arrhythmogenic right ventricular cardiomyopathy. Blood material was collected from scar areas by aspiration and filtered, CMs detected by light microscopy were aspirated, centrifugated, and freezed.DNA was extracted, amplified, and sequenced, and variants were compared with variants obtained from leukocyte DNA. RESULTS: At least 1 CM was obtained in 95% of patients (median 11 CM/patient). After refinements of the technique, a total of 136 samples (22 patients) allowed DNA extraction and amplification, successful in 60% of samples (16 patients). DNA capture sequencing of a panel of cardiomyopathy-associated genes was successfully performed in 14 patients and compared with blood sequencing in 11. After controlled by Sanger, an additional variant, not present in blood, has been confirmed in CM in one patient. CONCLUSION: This new mini-invasive technique of sampling allows to perform genetic analysis on CMs. Pending future improvements, this technique could provide new sources of human cells for research and potential mosaicism detection.

Abstract:
A hallmark of Alzheimer's disease (AD) is the accumulation of amyloid plaques, primarily composed of misfolded amyloid β (Aβ) peptides. Complementary high-resolution imaging techniques are employed to investigate the plaque penetrability and the extracellular space (ECS) rheology in a mouse model of AD. Two-photon shadow imaging in vivo confirms that a dense ring of cells surrounds cortical amyloid plaques but highlights the diffusional penetrability of the amyloid core. Quantum dot tracking unveils that ECS diffusional parameters are heterogeneous in and around plaques, with an elevated diffusivity within and around plaques compared to wild-type-tissue. The amyloid core shows low nanoparticle density, varying by plaque phenotype. Carbon nanotube tracking confirms these altered local rheological properties at the level of the whole cortex of AD mice. Finally, the extracellular matrix is found to be dysregulated within the amyloid plaque, which may account for the observed alterations in diffusivity. This study provides fresh insights for understanding Aβ plaque penetration, a prerequisite for therapeutic development.

Abstract:
While blood vessels and osteocytes have been studied independently, their simultaneous changes with age remain undescribed. Our objective was to investigate the age-related evolution of both osteocyte and blood vessel networks in mouse cortical bone, and to assess the associated effects on osteocyte markers and oxygen intracellular levels. We analyzed femurs of male Flk1-GFP mice from growing, mature, middle-aged, and aged groups with techniques such as laser microdissection followed by RT-qPCR, tissue clearing and 3D fluorescence imaging. In the mature animals - when the cortical bone was thicker than in the growing animals - the osteocyte density, the number of dendrites per osteocyte and the blood vessel density were lower. This was associated with a reduced expression of Pdpn and with a smaller fraction of osteocytes exhibiting low intracellular oxygen. In aged animals - when cortical bone was thinner than in mature animals - the number of dendrites per osteocyte and the blood vessel density were lower. This was associated with a reduced Gja1 (Cx43) expression. Our results suggest that changes in the osteocyte network during maturation and aging are led by distinct mechanisms, and that the cortical bone blood vessels are not the main source of oxygen for osteocytes.

Abstract:
Bronchial smooth muscle (BSM) remodeling is an important feature of severe asthma pathophysiology. We previously showed that asthmatic BSM is metabolically different and increased rhinovirus (RV) replication rate, the main trigger of severe asthma exacerbations. Extracellular vesicles (EVs) are the key mediator in cell-cell communication, but the role of BSM cells-derived EVs on bronchial epithelial has never been investigated in asthma. Using severe asthmatic and non-asthmatic tissue collection, we show that asthmatic BSM cells are able to produce a greater amount of EVs containing metabolites involved in bioenergetics. We study the bronchial epithelium energetic rewiring following stimulation with asthmatic BSM cells-derived EVs. Modifications of bronchial epithelium metabolic behavior were associated with an increased ATP production and a breakdown of bronchial epithelium barrier function such as ciliary beating frequency and efficiency. Finally, we show that asthmatic BSM cells-derived EVs increased RV replication in bronchial epithelium following RV infection.

Abstract:
Obesity-induced biological changes often persist after weight loss and are difficult to reverse, a phenomenon known as 'obesogenic memory'. This enduring effect is associated with metabolic inflammation, particularly in adipose tissue. In this study, we characterise a mouse model of obesogenic memory and evaluate the efficacy of the unimolecular conjugate GLP-1/Dexa, which selectively and safely delivers the anti-inflammatory drug dexamethasone to GLP-1 receptor (GLP-1R)-expressing cells. We document that this precision pharmacological approach outperforms treatment with GLP-1 or dexamethasone alone, significantly reducing body weight, food intake, adiposity and markers of adipose tissue inflammation in male mice with obesogenic memory. In addition, we identify the CCR2/CCL2 inflammatory pathway as an important mediator of glucose intolerance and adipose tissue inflammation associated with obesogenic memory. Our findings suggest that targeting inflammation via GLP-1R signalling may be a promising therapeutic strategy to alleviate obesogenic memory and improve the long-term clinical management of metabolic diseases.

Abstract:
Huntington's disease (HD) is a complex neurodegenerative disorder with cognitive and motor symptoms that typically manifest in adulthood. However, embryonic brain development impairments leading to cortical defects in HD mutation carriers has been shown recently supporting a neurodevelopmental component in HD. Despite HD is primarily recognized as a striatal pathology, developmental alterations in this structure, particularly during the early postnatal period, remain poorly understood. To fill this gap, we examined striatal development in newborn R6/1 mice. We found that D2 receptor-expressing indirect-pathway medium spiny neurons (D2-MSNs) present in the matrix striatal compartment undergo early morphological and electrophysiological maturation. Altered electrophysiological properties were also observed in newborn CAG140 mice. Additionally, we also observed a D2-MSN-selective reduction in glutamatergic cortico-striatal transmission at the beginning of the second postnatal week as well as a reduced projection of D2-MSNs onto the GPe at birth in R6/1 mice. All these alterations were transient with the circuit normalizing after the second postnatal week. These results identify a compartment- and cell-type specific defect in D2-MSNs maturation, which can contribute in their latter vulnerability, as this cell-type is the first to degenerate in HD during adulthood.