Abstract Background Red blood cells from individuals with type 2 diabetes (T2D-RBCs) have been shown to induce endothelial dysfunction, a novel phenomenon mediated by extracellular vesicles (EVs) released from RBCs that invade endothelial cells. However, the mechanisms regulating EV release and uptake, and their impact on endothelial function in T2D, remain largely unknown. Purpose This study aimed to elucidate the molecular mechanisms governing RBC-EV release and uptake, and to determine how these processes affect endothelial function in T2D. Methods RNA-sequencing was conducted on human RBCs for transcriptomic profiles. EVs were isolated with the ExoEasy kit and quantified by nanoparticle tracking analysis. Expression analysis was conducted by qPCR and immunofluorescence. EVs were incubated with isolated mouse aortas for endothelium-dependent relaxation (EDR) with a wire myograph. Loss of function was achieved with GapmeR injection in db/db mice. EV uptake was assessed in human carotid artery endothelial cells (HCtAECs) following incubation with DiL-labeled EVs. Results RNA-sequencing identified ATP9A, a lipid flippase, as one of the most upregulated transcripts in T2D-RBCs compared to healthy controls (Fig. 1A). Gene ontology analysis highlighted enrichment of EV-related pathways (Fig. 1B-C), suggesting dysregulated EV biology in T2D-RBCs. qPCR (Fig. 1D) and immunofluorescence confirmed significantly elevated ATP9A expression in both T2D-RBCs (Fig. 1E-F) and diabetic db/db mouse RBCs (Fig. 1G-F). Notably, T2D-RBCs (Fig. 1I) and db/db RBCs (Fig. 1J) released fewer EVs than healthy controls, and human RBC ATP9A levels inversely correlated with EV release from those RBCs (Fig. 1K-L). In vivo ATP9A knockdown by injection of a GapmeR targeting ATP9A in db/db mice did not affect glycemia or body composition but increased vascular oxidative stress and further impaired EDR (Emax: ~29.4% in db/db Gapmer compared to Emax: ~49% in db/db). In vivoGapmeR injection resulted in a significant reduction in mRNA and protein levels of ATP9A in db/db RBCs (Fig. 2A-B), which worsened EDR in wild-type aortas (Fig. 2C). Further, ex vivo GapmeR transfection in isolated db/db aortas had no effect on EDR, and T2D-RBCs did not alter ATP9A expression in wild-type aortas (data not shown). This suggests that the exacerbation of endothelial dysfunction observed upon ATP9A knockdown was mediated by RBCs rather than direct modulation of vascular ATP9A. The RBCs isolated from GapmeR injected db/db mice released higher amounts of EVs, that were not altered in size (Fig. 2D-E). These EVs caused further impairment in EDR (Fig. 2F) and showed increased uptake by HCtAECs (Fig. 2G-H). Conclusion Our findings indicate that ATP9A upregulation in RBCs may act as a compensatory mechanism to limit EV release and mitigate the extent of EV-driven endothelial dysfunction in T2D (Fig. 2I).Figure 1For image description, please refer to the figure legend and surrounding text. Figure 2For image description, please refer to the figure legend and surrounding text.
Humoud et al. (Fri,) studied this question.