Di-san junyi daxue xuebao (Apr 2020)
Effect of platelet microvesicles on the function of human microvascular endothelial cells
Abstract
Objective To investigate the effect of platelet microvesicles (PMVs) on the function of human microvascular endothelial cells (HMECs). Methods Blood samples from healthy volunteers were collected and anticoagulated with one-tenth volume of citrate solution to isolate platelet-rich plasma (PRP). PMVs released from adenosine diphosphate (ADP)-activated human platelets was collected by centrifugation. HMECs were cultured and grouped as following: Tyrode's buffer as control (Con) group, final concentration of PMVs at 10, 20, 50 μg/mL respectively, and supernatant (Sup) of PMVs (almost PMVs free) for 24 h. ELISA was used to detect the contents of thromboxane B2 (TXB2) and 6-keto-PGF1α, the stable metabolites of TXA2 and prostaglandin I2 (PGI2) respectively, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in the culture supernatant of HMECs. The expression of endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) phosphorylation in HMECs were determined by Western blotting. Nitric oxide (NO) production in HMECs was measured by fluorescent probe. Endothelial microvesicles (EMVs) secreted by HMECs in the culture supernatant were determined by flow cytometry. Results Compared with Con group and Sup group, PMVs significantly upregulated the level of TXB2 and decreased that of 6-keto-PGF1α in the supernatant of HMECs in a time- and dose-dependent manner (P < 0.05). After incubation with PMVs, the phosphorylated eNOS and the production of NO in HMECs were reduced, while the expression levels of ET-1, ICAM-1, VCAM-1 and E-selectin in the supernatant of HMECs were increased significantly (P < 0.05). The concentration of EMVs induced by 50 μg/mL PMVs for 24 h was were increased obviously when compared with con group and Sup group (P < 0.05). Conclusion PMVs may induce the to dysfunction and damage of microvascular endothelial cells. This might be one of the important mechanisms by which platelets are involved in microcirculation dysfunction.
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