Myeloperoxidase delivered by erythrocyte-derived extracellular vesicles promotes oxidative stress and endothelial dysfunction in type 2 diabetes

It was recently demonstrated that red blood cells (RBCs) from individuals with type 2 diabetes (T2D-RBCs) induce endothelial dysfunction through their extracellular vesicles (EVs). However, the underlying molecular mechanisms remain unclear. Myeloperoxidase (MPO), a pro-inflammatory enzyme, mediator of oxidative stress and cardiovascular injury, has been shown to be present in RBC membrane and in RBC-derived EVs, but its role in endothelial dysfunction mediated by RBC-derived EVs in T2D has not been explored.

We sought to determine whether RBC-derived MPO contributes to T2D-associated endothelial dysfunction through transfer by EVs.

RBCs isolated from individuals with T2D and age-matched healthy controls (H-RBCs) were incubated with Krebs-Henseleit buffer (20% haematocrit, 18h) to release EVs. Wild-type mouse aortae were incubated with H-RBCs, T2D-RBCs, or their EVs for 18h, and endothelium-dependent relaxation (EDR) was assessed using a wire myograph. To evaluate MPO involvement, a selective MPO inhibitor (CAS 5351-17-7) was applied during RBC- or EV-aortae co-incubation (100 µM) or post-incubation (5 µM). MPO activity and protein expression in RBCs, EVs, and aortae were measured using immunofluorescence and immunohistochemistry. Reactive oxygen species (ROS) levels were assessed in human endothelial cells after incubation with EVs.

Exposure of mouse aortae to T2D-RBCs significantly impaired EDR (Fig. 1A). MPO protein expression was increased in T2D-RBCs (Fig. 1B-C), and EDR impairment was attenuated by MPO inhibition (Fig. 1D). Incubation with T2D-RBCs upregulated MPO protein expression in the aortae (Fig. 1E-F). Accordingly, the impairment in EDR induced by T2D-RBCs was significantly attenuated by inhibition of MPO in the aortae (Fig. 1G). Oxidative stress, detected by 4-hydroxynonenal (4-HNE) staining, was higher in vessels exposed to T2D-RBCs, but MPO inhibition reduced this effect (Fig. 1H-I). Furthermore, EVs derived from T2D-RBCs (T2D RBC-EVs) exhibited elevated MPO protein expression (Fig. 2A-B) and activity (Fig. 2C), and also significantly impaired EDR (Fig. 2D). Co-incubation with an MPO inhibitor attenuated endothelial dysfunction caused by T2D RBC-EVs (Fig. 2E). Immunohistochemical analysis showed upregulated MPO in aortae after incubation with T2D RBC-EVs (Fig. 2F-G). Inhibition of vascular MPO prevented EDR impairment (Fig. 2H), increased 4-HNE expression in vascular tissue (Fig. 2I-J), and enhanced ROS production in human endothelial cells (Fig. 2K-L).

T2D-RBCs and their EVs induce endothelial dysfunction through the delivery of MPO and induction of MPO-driven vascular oxidative stress. Our findings reveal a previously unrecognized mechanism of RBC-induced vascular injury in T2D and identify RBC-derived MPO as a potential therapeutic target for preventing endothelial dysfunction 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.