Decreased ANGPTL4 impairs endometrial angiogenesis during peri‐implantation period in patients with recurrent implantation failure

Figures

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  Figure 1. Expression of ANGPTL4 and PPARγ in endometrial tissues and serum of RIF patients and fertile women. (A) Immunohistochemical staining of ANGPTL4 and PPARγ in endometrium. (B) The mRNA levels of ANGPTL4, PPARγ, IGFBP1and PRL in endometrial tissues of RIF patients (n = 18) and fertile women (n = 18). (C and D) Protein levels of ANGPTL4 and PPARγ in endometrial tissues (n = 18 for each group). (E) Concentrations of ANGPTL4 in the serum from RIF patients (n = 18) and control group (n = 18). (F) Correlation analysis between the mRNA and protein levels of ANGPTL4 and PPARγ in endometrial tissues among all subjects (n = 36). Scale bar = 100 μm. The data are shown as the mean ± SD values. *P < .05, **P < .01 and ***P < .001 compared with corresponding control. Blots are representative

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  Figure 2. Changes of IGFBP1, PRL, PPARγ and ANGPTL4 in hESCs after decidualization. (A) Cell morphologies of hESCs on day 0 and 4 after in vitro decidualization. (B) The mRNA levels of IGFBP1, PRL, PPARγ and ANGPTL4 in hESCs after in vitro decidualization. (C) Protein levels of PPARγ and ANGPTL4 in hESCs after in vitro decidualization. (D) Concentrations of ANGPTL4 in culture medium of hESCs after in vitro decidualization. The data are shown as the mean ± SD values of at least three independent experiments performed. *P < .05 and **P < .01 compared with corresponding control

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  Figure 3. PPARγ mediates rosiglitazone‐induced up‐regulation of ANGPTL4 in hESCs. Decidual cells were treated with rosiglitazone (0, 0.25, 0.5, 1, 10 and 100 μM) for 24 h with pre‐treatment of T0070907 (10 μM) or not. Effect of rosiglitazone on mRNA levels of (A) IGFBP1, (B) PRL, (C) PPARγ and (D) ANGPTL4 in decidual cells. (E‐G) Effect of rosiglitazone on protein abundance of PPARγ and ANGPTL4 in decidual cells. (H) Concentrations of ANGPTL4 in culture medium of decidual cells. The data are shown as the mean ± SD values of at least three independent experiments performed. *P < .05, **P < .01 and ***P < .001 compared with corresponding control

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  Figure 4. PPARγ mediates rosiglitazone‐induced up‐regulation of ANGPTL4 in HUVECs. HUVECs were treated with rosiglitazone (0, 0.25, 0.5, 1, 10 and100 μM) for 24 h with pre‐treatment of T0070907 (10 μM) or not. (A and B) Effects of rosiglitazone on mRNA levels of PPARγ and ANGPTL4 in HUVECs. (C‐E) Typical bands and statistical results of protein abundance of PPARγ and ANGPTL4 in HUVECs after rosiglitazone treatment. Arrowhead denotes the specific band of ANGPTL4 in HUVECs. (F) Concentrations of ANGPTL4 in culture medium of HUVECs after rosiglitazone treatment. The data are shown as the mean ± SD values of at least three independent experiments performed. *P < .05 and **P < .01 compared with corresponding control

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  Figure 5. ANGPTL4 mediates rosiglitazone‐induced HUVECs proliferation. (A‐C) mRNA levels, protein levels and concentrations of ANGPTL4 in culture medium after siRNA‐mediated knockdown of ANGPTL4 in HUVECs. (D) Cell counting Kit‐8 assay was performed to examine cell proliferation at the indicated time points on HUVECs after siRNA‐mediated knockdown of ANGPTL4 and followed by treatment of rosiglitazone (10 μM; 24h) or rhANGPTL4 (100 nM; 24h). NC, negative controls. The data are shown as the mean ± SD values of three independent experiments performed, and there are 6 wells for each group in CCK‐8 assay. *P < .05 and **P < .01 compared with corresponding control

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  Figure 6. Effect of Rosiglitazone and ANGPTL4 on migration and angiogenesis in HUVECs. Wound‐healing assays were performed to assess the migratory abilities in HUVECs after treatments described as in CCK‐8 assay. Tube formation assay was performed to observe angiogenesis in HUVECs after treatments. (A) Images were taken after cell scratching at 0 h, 24 h and 48 h respectively. (B) Wound closure rates were compared at 24 h and 48 h. (C) Images were taken 24 h after cell plating respectively. (D) Total tube length, number of branches and number of meshes were analysed. NC, negative controls. Scale bar = 100 μm or 200 μm. The data are shown as the mean ± SD values of three independent experiments performed. *P < .05, **P < .01 and ***P < .001 compared with corresponding control