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

Abstract

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. ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blotting. Cell proliferation, invasion, and apoptosis were determined using a 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 blotting. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using the TUNEL method. Expression of ANXA4, PI3K, p-Akt and p-eNOS was downregulated in human PE placentas and PE placenta-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. Furthermore, inhibition of the PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Furthermore, 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 by an increase in expression of PI3K, p-Akt, and p-eNOS in rat placentas. Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/Akt/eNOS pathway.

Integrated bioinformatics analysis reveals novel key biomarkers and potential candidate small molecule drugs in gestational diabetes mellitus

“…CEBPB (CCAAT enhancer binding protein beta) [38], ACSL4 [39], MBD2 [40], ULK1 [41], NUCB2 [42], TWIST1 [43], HOOK2 [44], CLDN7 [45], TBK1 [46], YIPF6 [47], TFRC (transferrin receptor) [48], ENPP2 [49], SLIT2 [50], MFGE8 [51], FAT1 [52], GPC4 [53], COL6A3 [54], EGFL6 [55], AOC3 [56], CCN2 [57], LYVE1 [58], RARA (retinoic acid receptor alpha) [59], COL18A1 [60], THY1 [61], CD36 [62], PEMT (phosphatidylethanolamine N-methyltransferase) [63], AIF1L [64], OXTR (oxytocin receptor) [65], LMNA (lamin A/C) [66], CXCL14 [67], DKK3 [68], ANGPTL2 [69] and CMTM7 [70] were reported to be associated with obesity, but these genes might be linked with progression of GDM. AHR (aryl hydrocarbon receptor) [71], STS (steroid sulfatase) [72], PLAC1 [73], CYP11A1 [74], PSG11 [75], STAT5B [76], TLR3 [77], FOLR1 [78], HSPB1 [79], HSP90AA1 [80], ANXA4 [81], ATF3 [82], DAPK1 [83], ENTPD1 [84], ABL1 [85], VSIG4 [86], CD99 [87], VWF (von Willebrand factor) [88], PODXL (podocalyxin like) [89], PDPN (podoplanin) [90], RND3 [91], VCAN (versican) [92], AXL (AXL receptor tyrosine kinase) [93], PIEZO1 [94], GAS6 [93], LAMA4 [95], CAV1 [96], DLL1 [97], CD44 [98], CD81 [99], SMAD3 [100], NES (nestin) [101], DCN (decorin) [102], AGTR1 [103], SLIT3 [104], B2M [105], STAT3 [106], STC1 [107] and ADAMTS1 [108] were shown to participate in facilitating the preeclampsia. Majchrzak-Celiń ka et al [109] ...…”

MicroRNAs in Uteroplacental Vascular Dysfunction

“…Like MAPK signaling pathway, the PI3K/AKT signaling pathway is also important for trophoblast invasion [227]. This pathway in trophoblasts appeared to be impaired in preeclampsia [228]. Both preeclamptic and IUGR placentas exhibited reduced expression of miR-16, miR-21, and miR-144 [61,72,113].…”

“…The increased expression of let-7d in preeclamptic placentas decreased both MMP2 and MMP9 expression and suppressed trophoblast migration, although it was not determined whether MMP2 and MMP9 were targets of let-7d [58]. Since MMP-2/9 expression is also regulated by various signaling pathways such as MAPK, PI3K/AKT, Nodal, Wnt and TGF-β signal pathways, miRNAs which disrupted these pathways could also indirectly impaired MMP-2/9 expression, leading to impaired trophoblast invasion [76,80,223,228,246,281,282].…”

“…All of them targeted NOS3 to inhibit both eNOS expression and migration/invasion of HTR-8/SVneo cells. The functional importance of eNOS was further confirmed by the observation that NOS3 knockdown diminished annexin A4 overexpression-induced promotion of cell invasion in both HTR-8/SVneo and JEG-3 cells [228]. Moreover, pregnancy induced less increase in radius of uterine arteries in an eNOS-deficient (eNOS -/- ) mouse model of IUGR [186,292].…”

Novel Epigenetic Biomarkers in Pregnancy-Related Disorders and Cancers

“…Previous studies emphasized the similarities between placental and tumor tissues and research mostly progressed in the direction of using the knowledge about trophoblast behavior to better understand tumor behavior [33,34]. In recent years, there have been more and more studies where a certain molecule found to be important for the behavior or targeting of tumor cells has subsequently been associated with the functioning of trophoblasts and gestational diseases [35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51]. To bring the reader closer to the premise that the same molecules can mediate tumor and trophoblast behavior, we will discuss some of these studies in the paragraph below.…”