Aims/hypothesis

A hallmark chronic complication of type 2 diabetes mellitus is vascular hyperpermeability, which encompasses dysfunction of the cerebrovascular endothelium and the subsequent development of associated cognitive impairment. The present study tested the hypothesis that during type 2 diabetes circulating small extracellular vesicles (sEVs) exhibit phenotypic changes that facilitate pathogenic disruption of the vascular barrier.

Methods

sEVs isolated from the plasma of a mouse model of type 2 diabetes and from diabetic human individuals were characterised for their ability to disrupt the endothelial cell (EC) barrier. The contents of sEVs and their effect on recipient ECs were assessed by proteomics and identified pathways were functionally interrogated with small molecule inhibitors.

Results

Using intravital imaging, we found that diabetic mice (Lepr^db/db) displayed hyperpermeability of the cerebrovasculature. Enhanced vascular leakiness was recapitulated following i.v. injection of sEVs from diabetic mice into non-diabetic recipient mice. Characterisation of circulating sEV populations from the plasma of diabetic mice and humans demonstrated increased quantity and size of sEVs compared with those isolated from non-diabetic counterparts. Functional experiments revealed that sEVs from diabetic mice or humans induced the rapid and sustained disruption of the EC barrier through enhanced paracellular and transcellular leak but did not induce inflammation. Subsequent sEV proteome and recipient EC phospho-proteome analysis suggested that extracellular vesicles (sEVs) from diabetic mice and humans modulate the MAPK/MAPK kinase (MEK) and Rho-associated protein kinase (ROCK) pathways, cell–cell junctions and actin dynamics. This was confirmed experimentally. Treatment of sEVs with proteinase K or pre-treatment of recipient cells with MEK or ROCK inhibitors reduced the hyperpermeability-inducing effects of circulating sEVs in the diabetic state.

Conclusions/interpretation

Diabetes is associated with marked increases in the concentration and size of circulating sEVs. The modulation of sEV-associated proteins under diabetic conditions can induce vascular leak through activation of the MEK/ROCK pathway. These data identify a new paradigm by which diabetes can induce hyperpermeability and dysfunction of the cerebrovasculature and may implicate sEVs in the pathogenesis of cognitive decline during type 2 diabetes.

Graphical Abstract

中文翻译：

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循环小细胞外囊泡介导糖尿病血管通透性过高

目标/假设

2 型糖尿病的一个标志性慢性并发症是血管渗透性过高，包括脑血管内皮功能障碍以及随后出现的相关认知障碍。本研究检验了以下假设：在 2 型糖尿病期间，循环的小细胞外囊泡 (sEV) 表现出表型变化，促进血管屏障的致病性破坏。

方法

从 2 型糖尿病小鼠模型和糖尿病人类个体的血浆中分离出的 sEV 具有破坏内皮细胞 (EC) 屏障的能力。通过蛋白质组学评估 sEV 的含量及其对受体 EC 的影响，并用小分子抑制剂对确定的途径进行功能性研究。

结果

通过活体成像，我们发现糖尿病小鼠 ( Lepr ^db/db ) 表现出脑血管通透性过高。将糖尿病小鼠的 sEV 静脉注射到非糖尿病受体小鼠后，血管渗漏增强。对糖尿病小鼠和人类血浆中循环 sEV 群体的表征表明，与从非糖尿病小鼠中分离出的 sEV 相比，sEV 的数量和大小有所增加。功能实验表明，来自糖尿病小鼠或人类的 sEV 通过增强细胞旁和跨细胞渗漏，诱导 EC 屏障快速而持续的破坏，但不会引起炎症。随后的 sEV 蛋白质组和受体 EC 磷酸蛋白质组分析表明，糖尿病小鼠和人类的细胞外囊泡 (sEV) 调节 MAPK/MAPK 激酶 (MEK) 和 Rho 相关蛋白激酶 (ROCK) 通路、细胞间连接和肌动蛋白动力学。这已被实验证实。用蛋白酶 K 处理 sEV 或用 MEK 或 ROCK 抑制剂预处理受体细胞可减少糖尿病状态下循环 sEV 的高通透性诱导作用。

结论/解释

糖尿病与循环 sEV 的浓度和大小显着增加有关。糖尿病条件下 sEV 相关蛋白的调节可以通过 MEK/ROCK 通路的激活诱导血管渗漏。这些数据确定了一种新的范例，通过这种范例，糖尿病可以诱发脑血管系统的渗透性过高和功能障碍，并可能表明 sEV 与 2 型糖尿病期间认知能力下降的发病机制有关。

图形概要