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