Abstract Background Type 2 diabetes (T2D) is a major risk factor for cardiovascular disease, with endothelial dysfunction as an early manifestation. Red blood cells (RBCs) have been recently recognised as important contributors to T2D-associated endothelial dysfunction through their extracellular vesicles (EVs). RBCs and RBC-derived EVs contain abundant and diverse non-coding (nc)RNAs, including circular(circ) and micro(mi)RNAs. However, whether RBC-derived ncRNAs are altered in T2D and contribute to endothelial dysfunction remain poorly understood. Purpose This study aims to elucidate how RBC-derived ncRNAs contribute to endothelial dysfunction in T2D. Methods RBCs were isolated from individuals with T2D (T2D-RBCs) and age-matched healthy controls (H-RBCs) and incubated with Krebs-Henseleit buffer for 18h. EVs were isolated from the conditioned medium using the ExoEasy kit. The ncRNA profile was identified by RNA sequencing and validated with qPCR and Sanger sequencing. The target protein regulated by ncRNAs was measured with immunohistochemistry. The miRNA targets that the circRNA can sponge were predicted in silico and confirmed by qPCR in RBCs and human carotid arterial endothelial cells (HCtAEC) following co-incubation with EVs. Endothelium-dependent relaxation (EDR) was assessed in rodent aortae incubated with human RBCs and their EVs using a wire myograph. Results RNA sequencing identified a novel RBC circRNA: circZC3H7A. The back-splicing junction of circZC3H7A was confirmed by Sanger sequencing (Fig. 1A-1B). qPCR revealed reduced circZC3H7A levels in RBCs from T2D individuals compared to healthy controls (Fig. 1C). A siRNA targeting the back-splice junction of circZC3H7A in H-RBCs increased vascular oxidative stress and impaired EDR (Fig. 1D). miR-591, predicted as a circZC3H7A target (Fig. 1E), was upregulated in T2D-RBCs (Fig. 1F), and its inhibition in T2D-RBCs improved EDR (Fig. 1G). Aortae incubated with T2D-RBCs exhibited reduced protein levels of miR-591 target: ZEB1 (Fig. 1H-1I), whereas inhibition of miR-591 in T2D-RBCs restored ZEB1 protein expression (Fig. 1J). miR-591 expression was increased in HCtAECs following co-incubation with EVs from T2D-RBCs compared to H-RBCs, even in condition when endogenous miR-591 in HCtAEC was knocked down prior to the co-incubation (Fig. 2A-2B), indicating that more T2D-RBC miR-591 is delivered via EVs to endothelial cells. Notably, aortae incubated with EVs from T2D-RBCs where miR-591 was inhibited improved EDR (Fig. 2C) and exhibited reduced protein levels of ZEB1 (Fig. 2C). Conclusions These findings suggest that downregulation of circZC3H7A in T2D-RBCs likely regulate miR-591 expression, and EV-mediated transfer of miR-591 to endothelial cells suppresses ZEB1, leading to endothelial dysfunction. Targeting RBC-derived ncRNAs may provide new strategies for preventing vascular injury in T2D.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.
Kontidou et al. (Fri,) studied this question.