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-enriched 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 BAT function. However, we also uncover that culturing BAT-ECs profoundly remodels EC identity. Transcriptomic profiling shows that BAT-ECs rapidly downregulate BAT-enriched endothelial markers and acquire features resembling WAT-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. • Fast method to purify endothelial cells (ECs) for direct RNA or protein extraction. • Easy method to isolate and culture ECs specifically from thermogenic adipose tissue. • Novel signature of BAT-ECs regulated upon browning and lost in culture. • RGC-32, p57 kip2 and EFNB1 as novel BAT-EC-enriched markers activated through ß-adrenergic stimulation.
Elschner et al. (Sun,) studied this question.