In the original publication [...].

Abstract Introduction Reliable generation of hepatocyte-like cells (HLCs) from pluripotent stem cells remains limited by heterogeneity and incomplete maturation of the cells. Derivation of induced pluripotent- and embryonic stem cells into hepatocytes typically relies on complex, and costly reagent-intensive protocols, with inconsistent reporting of differentiation efficiencies and functional maturation criteria. Variability in protocol designs highlights the need for optimisation, particularly in mouse embryonic stem cells (mESCs) systems that can be more comparable with mouse models for underpinning translational and toxicological studies. Here, we developed and evaluated two cytokine-based strategies: an advanced hepatic-inducing cocktail (A-HIC) and a simplified hepatic-inducing cocktail (HIC), both designed to reduce complexity while increasing functional maturation. Methods Hepatic differentiation and maturation were assessed by morphology, immunofluorescence, flow cytometry, and qRT-PCR. Functional competence was evaluated via urea production, glutathione synthesis, indocyanine green handling, cytochrome P450 inducibility, and impedance-based cell layer integrity monitoring. Results Morphological, molecular and phenotypic analyses confirmed that both protocols supported hepatic lineage progression, generating heterogeneous populations of hepatoblast-like and more mature HLCs. Gene expression confirmed the loss of pluripotency, transient endoderm induction, and subsequent hepatic specification. Functionally, cells exhibited glycogen storage, inducible urea production, glutathione depletion, and active ICG uptake and clearance, with stable monolayer formation by day 21. A-HIC-derived HLCs demonstrated enhanced maturation, with higher ASGR1 expression and stronger Cyp1a1 induction. Discussion These findings suggest that both protocols generate functional HLCs; however, A-HIC yields a higher proportion of functionally mature cells with reduced variability. This approach enables a simple, cost-effective, and time-efficient generation of HLCs, supported by improved functional characterisation with potential applicability to more complex pluripotent systems, including human iPSC-based models for disease modelling and toxicology.

We are delighted to share with you our fifteenth Journal Club and highlight some of the most interesting papers published recently [...].

Chronic wounds (CWs) represent a major clinical challenge, characterized by persistent inflammation and failed repair. While proresolving pathways are known to regulate inflammatory responses, their potential dysfunction in CWs remains unexplored. Here, we identify dysregulation of the pro-opiomelanocortin-melanocortin 1 receptor (POMC-MC1R) axis as a common feature across pressure ulcers, venous ulcers, and diabetic ulcers. Using MC1Re/e mice lacking functional MC1R, we demonstrate impaired wound healing marked by delayed reepithelialization and increased neutrophil extracellular traps-pathological features observed in human CWs. To investigate MC1R's therapeutic potential, we developed a new murine CW model that replicates human pathology, presenting as nonhealing, exudate-rich ulcers. Topical application of the MC1R-selective agonist BMS-470539 restored healing by reducing exudate production, stimulating vascularization, and enabling reepithelialization. The critical role of MC1R was further evidenced by MC1Re/e mice, which developed more severe ulcers with excessive exudate and NETosis. In acute wound studies, we found that topical MC1R agonist enhanced wound bed perfusion and lymphatic drainage through increased angiogenesis and lymphangiogenesis and reduced scarring by modulating fibroblast phenotype. Together, these findings establish the MC1R/POMC axis as a fundamental regulator of skin repair and identify promising therapeutic strategies to drive healing.

Aims Vascular calcification is the abnormal deposition of calcium phosphates within blood vessels. This condition is significantly associated with the development of cardiovascular disease, yet the underlying mechanisms remain largely unknown. MicroRNAs (miRNAs) may be crucial in initiating vascular calcification by regulating a network of specific cellular targets. In this study, we explored for the first time the potential role of microRNA-26b (miR-26b) in vascular calcification.Methods and results Using micro-positron emission tomography and computed tomography (micro-PET/CT) imaging with 18F-sodium fluoride, we measured aortic calcification in miR-26b knockout mice (miR-26bKO). We conducted bulk RNA sequencing (RNA-seq), single-cell RNA sequencing, and network analysis to identify cell-specific targets and the cellular complexity contributing to the observed phenotype. Additionally, we examined aortic tissues from patients with aortic aneurysm or valvular-related aortopathy to determine how the expression levels of miR-26b and its targets correlate with calcification. Our findings revealed that miR-26b is downregulated in the aortic tissues of patients with aortic calcification, whereas miR-26b expression negatively correlates with calcification levels. Similarly, miR-26bKO mice developed spontaneous age-related aortic microcalcifications. Combining single-cell transcriptomics with network analyses, we identified and mapped cell-type specific targets of miR-26b and regulatory pathways. Furthermore, we validated the cell-specific expression of Smad1 in smooth muscle cells (SMCs) and characterized the cell-cell communication between aortic cells, exposing the bone morphogenetic protein (BMP) pathway. The development of microcalcification was attributed to Bmp4 released from fibroblasts (FBLs), leading to Smad1 phosphorylation and calcium accumulation in SMCs of miR-26bKO mice. We found that aortic microcalcification could be pharmacologically reversed by disrupting cellular communication. Lastly, we demonstrated an inverse correlation between miR-26b and SMAD1 levels in calcified aortic tissues.Conclusion The deficiency of miR-26b is crucial for initiating and promoting aortic calcification, revealing new therapeutic targets for aortic disease.

Atherosclerosis is an underlying cause of cardiovascular diseases (CVD) which account for most deaths worldwide. Use of diverse preclinical models of atherosclerosis has been implemental in understanding the underlying mechanisms, the implicated cell types, the genes and the molecules at play in the onset and progression of atherosclerotic plaques. Although significant research advancements have been made, further research is necessary to delve into factors influencing plaque types, site preference within the vasculature, interactions with adjacent tissues (liver, pancreas and perivascular adipose tissue), inflammation and sex-based disparities, among others. The conventional low throughput methodologies which concentrate on individual cells, genes or metabolites are inadequate to tackle the complex and heterogeneous nature of atherosclerosis. With recent advancement in multi-omics and bioinformatics, research approaches have illuminated a clearer understanding of atherosclerosis. Consequently, these advancements pave the path to design novel therapeutics to complement currently approved lipid-lowering and other effective treatments. In this article, we summarize and critically evaluate the findings derived from recent high throughput single- or multi-omic studies conducted in animal models of atherosclerosis. We also delve into the challenges associated with using experimental animals to model human atherosclerosis and contemplate the essential enhancements needed to better mimic human conditions. We further discuss the requirement of establishing a structured multi-omic database for atherosclerosis research, enabling broader access and utilisation within the scientific community.

Systemic inflammation can lead to neuroinflammation with acute consequences such as delirium and long-lasting deleterious effects including cognitive decline and the exacerbation of neurodegenerative disease progression. Here, we show that transcription factor Nrf2 controls brain endothelial cell homeostasis and barrier strength. We found that peripheral inflammation caused infiltration of macrophages, microglial activation, and inflammatory reactive astrogliosis, all of which could be prevented by RTA-404, an activator of the transcription factor Nrf2 and close structural relative of the recently FDA-approved Nrf2 activator RTA-408 (omaveloxolone). To identify the key cellular mediator(s), we generated an endothelial cell-specific Nrf2 knockout mouse. Strikingly, the effects of RTA-404 on brain endothelial activation and downstream neuroinflammatory events were abolished by endothelial cell-specific Nrf2 deletion. This places endothelial cell Nrf2 as a peripherally accessible therapeutic target to reduce the CNS-adverse consequences of systemic inflammation.

The emergence of single-cell sequencing and computational analysis has dramatically improved our understanding of cellular diversity and gene expression dynamics. The rapid advancement of high-throughput omics technologies has led to an exponential growth in biological data. However, many gene regulatory processes at the single-cell level remain underexplored, especially those regulated by post-transcriptional mechanisms involving microRNAs (miRNAs). miRNAs are essential regulators of gene expression, affecting cellular functions in both normal and disease states. Recent innovations, such as single-cell gene expression profiling and bioinformatic analysis, have enabled comprehensive studies that uncover previously hidden miRNA profiles. In this context, we present experimental tools and computational methods for analysing cell-specific miRNA abundance and investigating their mechanisms. These approaches are expected to reveal the complex nature of miRNA biology and, more broadly, enhance our understanding of life sciences and diseases.

The involvement of the neurotrophin network in pathological skin conditions, such as psoriasis or squamous cancer, by their common neurotrophin receptor CD271 has become recently evident. Depending on the specific ligand and co-receptor interacting with it, CD271 mediates various cellular responses in keratinocytes. In vitro analysis shows that it is implicated in the transition from human interfollicular keratinocyte stem cells to transient amplifying cells. However, no in vivo models are available to dissect the complexity of these mechanisms, including the effect on the inflammatory response. Here, we develop and characterize two novel mouse models, the CD271cKO and the CD271ciKO, where CD271 is conditionally absent in keratinocytes during development or after topical induction, respectively. By histology, functional assay, transcriptomics and molecular analysis, we identified substantial skin changes correlated to CD271 deletion, including epidermal hyperproliferation, “activated” keratinocyte signature, and a delayed in the differentiation process, mostly linked to PI3K/Akt and mitogenic pathways-dependent processes. KO keratinocyte displays upregulation of Ki67, PCNA, KRT5, KRT6, and ERK phosphorylation, as well as major expression of IL1α, Cxcl15, and TGFβ. KO skin resemble dysplastic skin conditions, including the recruitment of immune cells, particularly T cells, macrophages, and neutrophils, and release of inflammatory cytokines involved in TNF, JAK/Stat, IL17, and PI3k/Akt signaling pathways. Overall, our data defines CD271 as a crucial regulator of skin homeostasis. Therefore, our models represent an exceptionally useful tool for the characterization of skin pathophysiology linked to CD271 and possibly for developing appropriate therapies.

The field of non-coding RNA research is advancing at a breathtaking pace, continually uncovering new layers of regulatory complexity and functional diversity [...]

Despite the discovery and prevalent clinical use of potent lipid-lowering therapies, including statins and PCSK9 inhibitors, cardiovascular diseases (CVD) caused by atherosclerosis remain a large unmet clinical need, accounting for frequent deaths worldwide. The pathogenesis of atherosclerosis is a complex process underlying the presence of modifiable and non-modifiable risk factors affecting several cell types including endothelial cells (ECs), monocytes/macrophages, smooth muscle cells (SMCs) and T cells. Heterogeneous composition of the plaque and its morphology could lead to rupture or erosion causing thrombosis, even a sudden death. To decipher this complexity, various cell model systems have been developed. With recent advances in systems biology approaches and single or multi-omics methods researchers can elucidate specific cell types, molecules and signalling pathways contributing to certain stages of disease progression. Compared with animals, in vitro models are economical, easily adjusted for high-throughput work, offering mechanistic insights. Hereby, we review the latest work performed employing the cellular models of atherosclerosis to generate a variety of omics data. We summarize their outputs and the impact they had in the field. Challenges in the translatability of the omics data obtained from the cell models will be discussed along with future perspectives.

Individuals diagnosed with Parkinson’s disease (PD) often exhibit heightened susceptibility to cardiac dysfunction, reflecting a complex interaction between these conditions. The involvement of mitochondrial dysfunction in the development and progression of cardiac dysfunction and PD suggests a plausible commonality in some aspects of their molecular pathogenesis, potentially contributing to the prevalence of cardiac issues in PD. Mitochondria, crucial organelles responsible for energy production and cellular regulation, play important roles in tissues with high energetic demands, such as neurons and cardiac cells. Mitochondrial dysfunction can occur in different and non-mutually exclusive ways; however, some mechanisms include alterations in mitochondrial dynamics, compromised bioenergetics, biogenesis deficits, oxidative stress, impaired mitophagy, and disrupted calcium balance. It is plausible that these factors contribute to the increased prevalence of cardiac dysfunction in PD, suggesting mitochondrial health as a potential target for therapeutic intervention. This review provides an overview of the physiological mechanisms underlying mitochondrial quality control systems. It summarises the diverse roles of mitochondria in brain and heart function, highlighting shared pathways potentially exhibiting dysfunction and driving cardiac comorbidities in PD. By highlighting strategies to mitigate dysfunction associated with mitochondrial impairment in cardiac and neural tissues, our review aims to provide new perspectives on therapeutic approaches.

The adult heart is a complex, multicellular organ that is subjected to a series of regulatory stimuli and circuits and has poor reparative potential. Despite progress in our understanding of disease mechanisms and in the quality of health care, ischaemic heart disease remains the leading cause of death globally, owing to adverse cardiac remodelling, leading to ischaemic cardiomyopathy and heart failure. Therapeutic targets are urgently required for the protection and repair of the ischaemic heart. Moreover, personalized clinical biomarkers are necessary for clinical diagnosis, medical management and to inform the individual response to treatment. Non-coding RNAs (ncRNAs) deeply influence cardiovascular functions and contribute to communication between cells in the cardiac microenvironment and between the heart and other organs. As such, ncRNAs are candidates for translation into clinical practice. However, ncRNA biology has not yet been completely deciphered, given that classes and modes of action have emerged only in the past 5 years. In this Review, we discuss the latest discoveries from basic research on ncRNAs and highlight both the clinical value and the challenges underscoring the translation of these molecules as biomarkers and therapeutic regulators of the processes contributing to the initiation, progression and potentially the prevention or resolution of ischaemic heart disease and heart failure.

Vascular permeability is temporarily heightened during inflammation, but excessive inflammation-associated microvascular leakage can be detrimental, as evidenced in the inflamed lung. Formylated peptides regulate vascular leakage indirectly via formylated peptide receptor-1 (FPR1)-mediated recruitment and activation of neutrophils. Here we identify how the GTPase-activating protein ARAP3 protects against formylated peptide-induced microvascular permeability via endothelial cells and neutrophils. In vitro, Arap3-/- endothelial monolayers were characterised by enhanced formylated peptide-induced permeability due to upregulated endothelial FPR1 and enhanced vascular endothelial cadherin internalisation. In vivo, enhanced inflammation-associated microvascular leakage was observed in Arap3-/- mice. Leakage of plasma protein into the lungs of Arap3-/- mice increased within hours of formylated peptide administration. Adoptive transfer experiments indicated this was dependent upon ARAP3 deficiency in both immune and non-immune cells. Bronchoalveolar lavages of formylated peptide-challenged Arap3-/- mice contained neutrophil extracellular traps (NETs). Pharmacological inhibition of NET formation abrogated excessive microvascular leakage, indicating a critical function of NETs in this context. The observation that Arap3-/- mice developed more severe influenza suggests these findings are pertinent to pathological situations characterised by abundant formylated peptides. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

EDITORIAL article Front. Cardiovasc. Med., 22 March 2024Sec. Cardiovascular Biologics and Regenerative Medicine Volume 11 - 2024 | https://doi.org/10.3389/fcvm.2024.1392073

Transplantation of adventitial pericytes (APCs) improves recovery from tissue ischemia in preclinical animal models by still unknown mechanisms. This study investigates the role of the adipokine leptin (LEP) in the regulation of human APC biological functions. Transcriptomic analysis of APCs showed components of the LEP signalling pathway are modulated by hypoxia. Kinetic studies indicate cultured APCs release high amounts of immunoreactive LEP following exposure to hypoxia, continuing upon return to normoxia. Secreted LEP activates an autocrine/paracrine loop through binding to the LEP receptor (LEPR) and induction of STAT3 phosphorylation. Titration studies using recombinant LEP and siRNA knockdown of LEP or LEPR demonstrate the adipokine exerts important regulatory roles in APC growth, survival, migration and promotion of endothelial network formation. Heterogeneity in LEP expression and secretion may influence the reparative proficiency of APC therapy. Accordingly, the levels of LEP secretion predict the microvascular outcome of APCs transplantation in a mouse limb ischemia model. Moreover, we found that the expression of the Lepr gene is upregulated on resident vascular cells from murine ischemic muscles, thus providing a permissive milieu to transplanted LEP-expressing APCs. Results highlight a new mechanism responsible for APC adaptation to hypoxia and instrumental to vascular repair.

Epigenetic processes involving long non -coding RNAs regulate endothelial gene expression. However, the underlying regulatory mechanisms causing endothelial dysfunction remain to be elucidated. Enhancer of zeste homolog 2 (EZH2) is an important rheostat of histone H3K27 trimethylation (H3K27me3) that represses endothelial targets, but EZH2 RNA binding capacity and EZH2:RNA functional interactions have not been explored in post-ischemic angiogenesis. We used formaldehyde/UV-assisted crosslinking ligation and sequencing of hybrids and identi fi ed a new role for maternally expressed gene 3 (MEG3). MEG3 formed the predominant RNA:RNA hybrid structures in endothelial cells. Moreover, MEG3:EZH2 assists recruitment onto chromatin. By EZH2chromatin immunoprecipitation, following MEG3 depletion, we demonstrated that MEG3 controls recruitment of EZH2/ H3K27me3 onto integrin subunit alpha4 ( ITGA4 ) promoter. Both MEG3 knockdown or EZH2 inhibition (A-395) promoted ITGA4 expression and improved endothelial cell migration and adhesion to fi bronectin in vitro . The A-395 inhibitor redirected MEG3 -assisted chromatin remodeling, offering a direct therapeutic bene fi t by increasing endothelial function and resilience. This approach subsequently increased the expression of ITGA4 in arterioles following ischemic injury in mice, thus promoting arteriogenesis. Our fi ndings show a context -speci fi c role for MEG3 in guiding EZH2 to repress ITGA4 . Novel therapeutic strategies could antagonize MEG3:EZH2 interaction for pre -clinical studies.