Wilhelm-Juengling, K. | bioRxiv Science

bioRxiv2025

From Brown to White: Brown Adipose Tissue Endothelial Cells whiten in Culture Conditions

Endothelial cells (ECs) are central regulators of vascular and metabolic homeostasis, yet their organ- and depot-specific diversity remains underexplored. Two major types of adipose tissue (AT) can be distinguished that differ substantially in their physiological function and vascularization: white AT (WAT), which is the major energy storage and brown AT (BAT), which is highly vascularized and dissipates energy [1-5]. While ECs from these depots likely contribute to adipose function, their characterization has been hindered by technical limitations in isolation and culture. Here, we establish a protocol for isolating and expanding ECs from murine BAT and WAT, enabling transcriptomic and functional analyses across depots. We demonstrate that freshly isolated BAT-ECs express depot-specific gene signatures, including Rgcc, Cdkn1c, Tcf15, Meox2, and Efnb1, several of which are dynamically regulated during cold-induced BAT activation. These findings reveal novel BAT-EC markers and highlight specialized endothelial programs that may support thermogenesis. However, we also uncover that culturing BAT-ECs profoundly remodels EC identity. Transcriptomic profiling shows that BAT-ECs rapidly downregulate BAT-specific markers and acquire features resembling WAT- and lymphatic ECs. This dedifferentiation is accompanied by signatures of proliferation, adhesion remodeling, and endothelial-to-mesenchymal transition. While these changes present challenges for maintaining depot-specific identity in culture, they also provide a framework to better interpret experimental outcomes and to investigate EC plasticity. Taken together, our study delivers a novel isolation and culture protocol for adipose ECs, defines BAT-EC markers, and demonstrates how culture conditions reshape their identity. These insights build the foundation for future research of AT vasculature. HighlightsO_LIFast method to purify endothelial cells (ECs) for direct RNA or protein extraction. C_LIO_LIEasy method to isolate and culture ECs specifically from thermogenic adipose tissue. C_LIO_LINovel signature of BAT-ECs regulated upon browning and lost in culture. C_LIO_LIRGC-32, p57kip2, TCF15/MEOX2 and EFNB1 as novel BAT-EC-specific markers. C_LI

cell biology↗

bioRxiv2025

Shiny-Calorie: A context-aware application for indirect calorimetry data analysis and visualization using R

Indirect calorimetry is a cornerstone technique for metabolic phenotyping of animal models in preclinical research, with well-established experimental protocols and platforms. However, a flexible, extensible, and user-friendly software suite that enables standardized integration of data and metadata from diverse metabolic phenotyping platforms--followed by unified statistical analysis and visualization--remains absent. We present Shiny-Calorie, an open-source interactive web application for transparent data and metadata integration, comprehensive statistical data analysis, and visualization of indirect calorimetry datasets. Shiny-Calorie is compatible with data formats from widely used commercial metabolic phenotyping platforms, such as TSE and Sable Systems, and includes functionality for exporting processed data in these formats. Built using GNU R and a Shiny-based reactive interface, Shiny-Calorie enables intuitive exploration of complex, multi-modal longitudinal datasets comprising categorical, continuous, ordinal, and count variables. The platform incorporates state-of-the-art statistical methods for robust hypothesis testing, thereby facilitating biologically meaningful interpretation of energy metabolism phenotypes, including resting metabolic rate and energy expenditure. Overall, Shiny-Calorie streamlines routine analysis workflows and enhances reproducibility and transparency in metabolic phenotyping studies.

bioinformatics↗

bioRxiv2022

Aortic valve disease augments vesicular microRNA-145-5p to regulate the calcification of valvular interstitial cells via cellular crosstalk

RationaleAortic valve stenosis (AVS) is a major contributor to cardiovascular death in the elderly population worldwide. MicroRNAs (miRNAs) are highly dysregulated in patients with AVS undergoing surgical aortic valve replacement (SAVR). However, miRNA-dependent mechanisms regulating inflammation and calcification or miRNA-mediated cell-cell crossstalk during the pathogenesis of AVS are still poorly understood. Here, we explored the role of extracellular vesicles (EV)-associated miR-145-5p, which we showed to be highly upregulated upon valvular calcification in AVS in mice and humans. MethodsHuman TaqMan miRNA arrays identified dysregulated miRNAs in aortic valve tissue explants from AVS patients compared to non-calcified valvular tissue explants of patients undergoing SAVR. Echocardiographic parameters were measured in association with the quantification of dysregulated miRNAs in a murine AVS model. In vitro calcification experiments were performed to explore the effects of EV-miR-145-5p on calcification and crosstalk in valvular cells. To dissect molecular miRNA signatures and their effect on signaling pathways, integrated OMICS analyses were performed. RNA sequencing (RNA-seq), high-throughput transcription factor (TF) and proteome arrays showed that a number of genes, miRNAs, TFs, and proteins are crucial for calcification and apoptosis, which are involved in the pathogenesis of AVS. ResultsAmong several miRNAs dysregulated in valve explants of AVS patients, miR-145-5p was the most highly gender-independently dysregulated miRNA (AUC, 0.780, p-value, 0.01). MiRNA arrays utilizing patient-derived- and murine aortic-stenosis samples demonstrated that the expression of miR-145-5p is significantly upregulated and correlates positively with cardiac function based on echocardiography. In vitro experiments confirmed that miR-145-5p is encapsulated into EVs and shuttled into valvular interstitial cells. Based on the integrated OMICs results, miR-145-5p interrelates with markers of inflammation, calcification, and apoptosis. In vitro calcification experiments demonstrated that miR-145-5p regulates the ALPL gene, a hallmark of calcification in vascular and valvular cells. EV-mediated shuttling of miR-145-5p suppressed the expression of ZEB2, a negative regulator of the ALPL gene, by binding to its 3 untranslated region to inhibit its translation, thereby diminishing the calcification of target valvular interstitial cells. ConclusionElevated levels of pro-calcific and pro-apoptotic EV-associated miR-145-5p contribute to the progression of AVS via the ZEB2-ALPL axis, which could potentially be therapeutically targeted to minimize the burden of AVS. Clinical SignificanceO_ST_ABSWhat is known?C_ST_ABSO_LIAortic valve stenosis (AVS) is the most prevalent structural heart valve disease requiring surgical or interventional valve replacement. Currently, no medical treatment option is available to slow, halt, or reverse the progression of the disease. C_LIO_LIAVS induces pressure overload on the left ventricle (LV), resulting in concentric hypertrophy and LV dysfunction. C_LIO_LIAVS is not an exclusively degenerative disease that leads to fibrosis and calcification of the valve cusps but rather a chronic inflammatory disease, in which mechanical strain and shear stress lead to endothelial dysfunction and immune cell infiltration, which induces chronic inflammation, apoptosis and differentiation of valvular interstitial cells into osteoblast-like cells. C_LIO_LIIncreasing osteoblastic differentiation and the formation of macrocalcifications are hallmarks of the later stages of AVS. C_LI What is the new information we provide?O_LIDuring aortic valve stenosis, expression pattern of vesicle-associated regulatory miRNAs is altered. C_LIO_LIPatient-derived aortic valve tissue demonstrated an increased expression of miR-145-5p in humans, as well as in aortic valve explants from an experimental murine AVS model. C_LIO_LIMiR145-5p contributes to calcification of the aortic valve through ZEB2, a transcriptional repressor of ALPL, in valvular interstitial cells. C_LIO_LIExtracellular vesicular shuttling of miR-145-5p contributes to valvular cell-cell crosstalk and plays a role in the pathogenesis of AVS. C_LI

cell biology↗