Abstract 13966: The Effect of Diabetes-Associated Adiposomes in Promoting Caveolar Loss and Endothelial Dysfunction

Introduction: Our recent findings revealed that ex vivo cultured human adipose tissues (AT) release extracellular vesicles (adiposomes) that are efficiently captured by endothelial cells. These adiposomes are lipid-enclosed structures that carry bioactive cargos and mediate signaling between the AT and remote cells and tissues. Under hyperglycemia, ceramides could be glycosylated into glycosphingolipids (GSLs), selectively packed in adiposomes to be cleared away from adipocytes. Hypothesis: We hypothesize that the AT from obese, diabetic patients (OB-T2D) creates excess GSL-rich adiposomes, which are incorporated into endothelial cells causing alterations in endothelial cell properties and function. Methods: AT samples were collected from OB-T2D and non-OB subjects (n=10, each). Nanoparticle tracking and electron microscopy were used to analyze and visualize these adiposomes, and mass spectrometry was used to study their content. Adiposome uptake, lipid fusion, and changes in endothelial membrane structure and signaling pathways were also assessed. Results: Compared to non-obese controls, adiposomes isolated from OB-T2D were higher in number and contained higher levels of GSLs. When added to cultured endothelial cells, GSL-rich adiposomes tended to accumulate in an endothelial structure known as caveolae, which concentrate signaling molecules such as endothelial nitric oxide synthase (eNOS) and others. Electron microscopy showed a caveolar loss from the cell surface, and live TIRF microscopy indicated increased caveolar detachment. We observed induced phosphorylation of the key caveolar protein, caveolin-1, by GSL-rich adiposomes known to cause caveolar fission. This effect was mediated via activation of Src kinase (induced phosphorylation), as it was inhibited by the Src kinase inhibitor, PP2. These molecular events were accompanied by reductions in endothelial nitric oxide synthase (eNOS) activation and nitric oxide production, disturbances in endothelial gap junction, and increased permeability. Conclusion: we conclude that dysfunctional AT in OB-T2D produces GSL-loaded adiposomes that fuse with endothelial cells and activate the Src kinase pathway resulting in caveolar fission and endothelial cell dysfunction