C-Reactive Protein-Induced Endothelial Microparticle Generation in HUVECs Is Related to BH4-Dependent NO Formation

Abstract

Background: C-reactive protein (CRP) has been proven to facilitate endothelial injury via reduced NO production. Endothelial microparticles (EMPs) have emerged as a novel marker of endothelial injury. Methods: In vitro cultured human umbilical vein endothelial cells (HUVECs) were incubated with CRP (20 mg/l) for 24 h. The numbers of EMPs with CD31- and CD51-positive staining were assessed flow-cytometrically, and NO production was measured using the Griess reaction in the presence or absence of tetrahydrobiopterin (BH₄), respectively. Results: The number of EMPs was significantly increased in HUVECs stimulated by CRP compared with the control group and, in parallel, NO production was decreased (p < 0.05). In the presence of CRP, pretreatment with BH₄ decreased EMP counts and restored NO production to baseline levels (p < 0.05) while pretreatment with 2,4-diamino-6-hydroxypyrimidine (DAHP), a BH₄ synthesis inhibitor, further prompted EMP formation and decreased NO production (p < 0.05). However, adding exogenous BH₄ after pretreatment with DAHP suppressed EMP formation and restored NO production (p < 0.05). Conclusions: This study demonstrates that CRP induces EMP generation in HUVECs and this effect is, at least in part, related to impaired BH₄-dependent NO production. Augmented EMP generation in HUVECs is suggested as a novel potential mechanism contributing to the pathogenesis of vascular injury related to CRP.