ANXA4 promotes trophoblast invasion via the PI3K/AKT/eNOS pathway in preeclampsia.

OBJECTIVES: The inadequate trophoblast invasion is associated with the development of preeclampsia (PE). Considering that annexin A4 (ANXA4) enhances tumor invasion, we aimed to explore the functional role of ANXA4 in trophoblast cells and to examine the underlying mechanism.

METHODS: ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blot. Cell proliferation, invasion, and apoptosis were determined using MTT assay, Transwell assay, and flow cytometry, respectively. The expression levels of matrix metalloproteinase (MMP)‑2, MMP‑9, phosphoinositide 3‑kinase (PI3K), AKT, phosphorylated (p)‑AKT, and phosphorylated-endothelial nitric oxide synthase (p-eNOS) were detected by Western blot. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using TUNEL method.

RESULTS: Expression of ANXA4, PI3K, p-AKT and p-eNOS were downregulated in human PE placentas and PE placentas-derived extravillous cytotrophoblasts (EVCTs). Furthermore, ANXA4 overexpression promoted cell proliferation and invasion, inhibited cell apoptosis, and upregulated protein expression of PI3K, p-AKT and p-eNOS in human trophoblast cells HTR-8/SVneo and JEG-3. By contrast, ANXA4 knockdown exerted the opposite effects. Moreover, inhibition of PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Moreover, eNOS knockdown abrogated the ANXA4 overexpression-induced promotion of cell invasion and MMP2/9 expression. Additionally, in N-nitro-L-arginine methyl ester (L-NAME)-induced PE rats, ANXA4 overexpression alleviated PE progression, accompanied with an increase in expression of PI3K, p-AKT and p-eNOS in rat placentas.

CONCLUSION: Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/AKT/eNOS pathway.