RP105 ameliorates hypoxia/reoxygenation injury in cardiac microvascular endothelial cells by suppressing TLR4/MAPKs/NF-B signaling

The radioprotective 105 kDa protein (RP105) has been implicated in the pathological process of multiple cardiovascular diseases through its functional and physical interactions with Toll-like receptor 4 (TLR4). However, the effects of RP105 on cardiac microvascular endothelial cells (CMECs) in response to hypoxia/reoxygenation (H/R) injury have not been extensively investigated. The aim of the present study was to elucidate the potential roles of RP105 in the protection of CMECs against H/R injury, and investigate the underlying mechanisms. CMECs isolated from Sprague-Dawley rats were transduced with adenoviral vectors encoding RP105 or green fluorescent protein (GFP). At 48 h post-transfection, CMECs were subjected to hypoxia for 4 h and reoxygenation for 2 h (H/R) to simulate the in vivo ischemia/reperfusion model. The mRNA and protein levels of RP105 were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The effects of RP105 on CMEC proliferation, migration and apoptosis were measured by GFP-8, Transwell chamber and flow cytometry assays, respectively. The secretion of interleukin (IL)-6 and tumor necrosis factor (TNF)- in the culture medium was measured by ELISA. Moreover, the expression level of TLR4, p38 mitogen-activated protein kinase (MAPK), extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, nuclear factor (NF)-B/p65, IL-6, TNF- and intercellular adhesion melecule-1 was evaluated by western blot analysis. The results demonstrated that RP105 was minimally expressed in CMECs subjected to H/R injury. Overexpression of RP105 via adenoviral vectors was able to significantly protect CMECs against H/R injury, as evidenced by the promotion of cell proliferation and migration, as well as the amelioration of inflammation and apoptosis. These beneficial effects were at least partly mediated through inhibition of TLR4/MAPKs/NF-B signaling. Therefore, RP105 may be a promising candidate for prevention against CMECs-associated H/R injury.