Abstract

Our previous study showed that circulating microvesicles (cMVs) of diabetic mice have negative effects on the function of endothelial progenitor cells (EPCs). Whether this is true in diabetic patients deserves further study. In this study, the effects of cMVs and EPC-derived MVs (EPC-MVs) on EPC migration, apoptosis, and reactive oxygen species (ROS) production in healthy controls, well-controlled, and uncontrolled diabetic patients were investigated. The levels of miR-126 and vascular endothelial growth factor receptor 2 (VEGFR2) in cMVs, EPC-MVs, and/or EPCs were analyzed. Moreover, miR-126 inhibitor or mimic was applied to EPCs to modulate the miR-126 level in EPC-MVs. We found the following: 1) the circulating EPC level was reduced but the circulating EPC-MV level increased in uncontrolled diabetic patients; 2) the cMVs and EPC-MVs of healthy controls had beneficial effects on EPCs (migration, apoptosis, ROS), whereas the effects were reversely changed in the cMVs and EPC-MVs of uncontrolled diabetic patients; and 3) the cMVs and EPC-MVs of uncontrolled diabetic patients carried less miR-126 and had downregulated VEGFR2 expression in EPCs. Manipulating the miR-126 level in EPC-MVs with inhibitor or mimic changed their function. The effects of cMVs and EPC-MVs are compromised in diabetes due to the reduction of their carried miR-126, which might provide a therapy target for diabetic vascular complications.

diabetes mellitus (DM) is considered one of the major risk factors for various cardiovascular complications. Endothelium dysfunction is a key initiator for vascular disease, which results from increased oxidative stress in the vascular cells (39). Endothelial progenitor cells (EPCs) are known to play important roles in maintaining vascular function and structure by repairing or replacing dysfunctional or injured endothelial cells (ECs) (33). Impaired EPC proliferation, differentiation, adhesion, mobilization, and survival have been reported in DM (1, 26). Our previous study has demonstrated that the circulating EPC level is decreased and the function of EPCs impaired in db/db diabetic mice (8). Moreover, we found that the circulating microvesicles (cMVs) of db/db diabetic mice compromised the functions of EPCs. Investigation on the regulatory effects of cMVs on EPCs in diabetic patients could provide novel therapeutic avenues for vascular complications of diabetes.

Extracellular MVs are submicrometric fragments released from the cells in response to activation and apoptosis (22, 37). cMVs are the MVs released from the cells in the blood and from the vascular wall. An elevation of cMV levels has been reported in vascular diseases such as thrombotic diseases, diabetes, and cardiovascular diseases (2, 8, 25). Furthermore, one study has shown that the level of cMVs could predict the severity of vascular diseases (30). MVs released from EPCs (EPC-MVs) have been reported to serve as an index for EPC loss and functional incompetence (27). Moreover, the level of circulating EPC-MVs can predict aortic stiffness in atherosclerotic patients (27). Therefore, circulating MV and EPC-MV levels could serve as biomarkers and predictors for vascular diseases. On the other hand, accumulating evidence suggests that MVs mediate cell-cell communication via transferring proteins, mRNAs, and miroRNAs (miRs) from their parent cells to the target cells (5, 17, 29). The functions of MVs are complex and multifactorial, depending on the stimulator and origin. Previously, we demonstrated that cMVs of diabetic mice impair the function of EPCs, whereas cMVs of healthy controls do not have detrimental effects on EPCs (8). However, whether this is true in diabetic patients remains unclear.

Previous studies have shown that miR-126 governs vascular integrity (35) and is a biomarker or mediator of vascular diseases (11, 32). Downregulation of miR-126 impairs EPC function (23). EPC-MVs have been shown to improve ischemia-reperfusion injury of hindlimb and kidney through the transfer of miR-126 to target cells (6, 28). In the meantime, miR-126 has been reported to regulate angiogenic process and EC/EPC function by modulating vascular endothelial growth factor receptor 2 (VEGFR2) (13, 15). Our previous study found that EPC-MVs affect EC functions and apoptosis via their carried miR-126 (34). However, it is unknown whether EPC-MVs, as one type of MVs, would affect EPC functions through the miR-126 and its downstream VEGFR2 pathway.

In the present study, we determined the effects of cMVs and EPC-MVs of diabetic patients on EPC survival and functions and explored whether miR-126/VEGFR2 is involved in the mechanism.

Circulating microvesicles (cMVs) are the extracellular MVs released from the cells in the blood and on the vascular wall. Our previous study demonstrates that cMVs of diabetic mouse are detrimental to endothelial progenitor cells (EPCs), which are known to be very important for maintaining normal endothelial function and structure. In this study, we compared the levels of circulating EPCs and EPC-derived MVs (EPC-MVs) in diabetic and healthy subjects. Also, the migration ability, apoptosis rate and reactive oxygen species (ROS) production of EPCs cultured from diabetic and healthy subjects were determined. More importantly, we evaluated whether cMVs from healthy subjects (ch-MVs) improves the function of EPCs from diabetic patients (d-EPCs), and whether cMVs from diabetic patients (cd-MVs) impairs the function of EPCs from healthy subjects (h-EPCs). The d-EPCs or h-EPCs were incubated with ch-MVs or cd-MVs for 24 hours. The migration ability of EPCs was analyzed by an assay kit. The apoptotic rate and ROS production were analyzed by labeling with propidium iodide (PI) and dihydroethidium (DHE) respectively, followed with flow cytometeric analysis. Our data showed that (1) there was a decrease in EPCs iv and an elevation in EPC-MVs in diabetic patients when compared to healthy subjects; (2) The migration ability of d-EPCs were decreased, and the apoptosis rate and ROS production were increased in d-EPCs; (3) ch-MVs improve the function of d-EPC through improving its migration ability and decreasing the apoptosis and ROS production; (4) cd-MVs increase h-EPC apoptosis, and increase ROS production. We conclude that cMVs modulate EPC function and this role of cMVs is reversed in diabetes with the mechanism linked to ROS production.