Fig 2: Schematic diagram shows the proposed mechanism of DICER mediating the invasion ability of HTR8 cells and the crosstalk between HTR8 cells and HUVECs during spiral artery remodelling in an in vitro model. DICER plays an important role in balancing the expression of miR-16-2 and let-7 family, and there seems to be a positive feedback between DICER and let-7 family where DICER inhibits the transcription of MIRLET7A1 gene and DICER is a target of let-7a (49). Increased level of miR-16-2-3p leads to decreased invasion ability and VEGFA secretion by targeting COL1A2 and VEGFA, respectively, in HTR8 cells following DICER knockdown. Silencing of DICER also creates an anti-angiogenic bias in the extracellular microenvironment of HTR8 cells with dysregulated angiogenic factors (up-regulated IGFBP3, down-regulated VEGFA, HB-EGF, MMP9 and ANGIOGENITIN), and increased miRNAs (miR-16-2, miR-15b and let-7d) and decreased DICER protein in HTR8 cell-derived MVs, leading to impaired angiogenesis of HUVECs

Fig 3: Effects of GDF11 in viability of MSCs in vitro. MSCs were cultured under serum deprivation and hypoxic conditions (0.1% O2) for 48 h to induce apoptosis in vitro. A, Viability of MSCs was examined by cell counting kit-8 (CCK-8) assay (n = 5). B, Apoptotic cells were detected with flow cytometry assay after staining with Annexin V & PI. C, Quantifications of apoptotic cells by adding Q2 and Q3 in B. D, TUNEL assay of apoptotic MSCs (pink). Nuclear were stained with Hoechst (blue). E, Quantifications of apoptotic MSCs in D (n = 3 in each group). F, Apoptosis-related proteins B-cell lymphoma-2 protein (BCL-2) and Bcl2 Associated X Protein (BAX) were detected by Western blot. G, Quantifications of BCL-2 and BAX related to ß-Actin (n = 3). H, mRNA levels were examined by RT-PCR for MSCs either transfected with NC-siRNA (control) or GDF11-siRNA (n = 3). I, Western blot assay for detection of proteins: GDF11, VEGFA and HGF after siRNA transfection. J, Quantitative analysis of I. K, Representative images of the cell morphology after transfected with specified siRNA for 24 h. Scale bars: 200 µm. L, Viability of MSCs was examined by CCK-8 assay after knock-down of GDF11 by siRNA (n = 5). M, Western blot assay for detection of apoptosis-related proteins BCL-2 and BAX after siRNA transfection. N, Quantitative analysis of M. Data are presented as mean ± SD for at least 3 independent experiments. *P < 0.05; **P < 0.01 and ***P < 0.001

Fig 4: The effect of GDF11 in differentiation of MSCs induced with VEGF165. A, Immunofluorescent staining of MSCVector (2nd raw), MSCGDF11 (3rd raw) with Abs against EC marker CD31 (red) and VEGFR2 (green). Nucleus was stained with DAPI (blue). MSCs were cultured at the presence of VEGF165 for 14 d to induce differentiation. Scale bars = 100 µm. HUVEC was used as positive control (3rd raw), MSCcontrol (1st raw) were cultured without VEGF165, and used as a negative control. B, Quantification of CD31+ cells in MSCVector and MSCGDF11 shown in A. C, Flow cytometry analysis of EC markers (CD31 in upper panel, VEGFR2 in lower panel) on MSCGDF11 and MSCVector. And HUVEC was used as positive control, MSCcontrol without VEGF induction were used as negative control. D, Quantification of positive rates in C (n = 3). E, Representative images showing tube formation of MSCs after differentiation for 14 d on Matrigel for 2 or 8 h. Scale bars: 200 µm. F, Quantification of tube formation in E by measuring branch lengths of formed tube for 8 h. Only length > 200 µm was counted (n = 5 in each group). Data are presented as the mean ± SD for at least 3 independent experiments. G, mRNA levels were examined by RT-PCR after cultured at the presence of VEGF165 for 14 days to induce differentiation for MSCs either transfected with NC-siRNA (control) or GDF11-siRNA (n = 3). H) Western blot assay for detection of proteins: GDF11, VEGFA, CD31, VEGFR1 and VEGFR2 after siRNA transfection and cultured with VEGF165 for 14 days. I, Quantitative analysis of H. *P < 0.05; **P < 0.01 and ***P < 0.001

Fig 5: Effect of GDF11 on the differentiation of MSCs into endothelial-like cells was blocked by ERK inhibitor. MSCVector and MSCGDF11 were treated with inhibitors for ERK (SD5978) or for TGF-ß receptor (LY2109761) or with DMSO as a control during the differentiation of MSCs into endothelial-like cells for 14 d. A, Representative results of flow cytometry analysis of MSCVector (Vector) and MSCGDF11 (GDF11) for their expression of CD31 and VEGFR2. B, Quantitative analysis of A (n = 3). C, Immunofluorescent staining of MSCVector and MSCGDF11 with antibody against CD31 (red) after culture with or without ERK inhibitor. Nucleus was stained with Hochest. Scale bars = 100 µm. D, Quantitative analysis of C (n = 3). CD31+ rate was obtained by dividing CD31+ cells (red) by total number of cells (blue) in a picture. E, Western blot analysis of proteins in MSCVector and MSCGDF11 in the presence of absence of inhibitors LY2109761 or SD5978during the differentiation of MSCs into endothelial-like cells. F, Quantitative analysis of E (n = 2). Each WB was repeated for at least 2 times. G, Schematic diagram of the proposed mechanisms by which GDF11 promotes MSC differentiation into endothelial-like cells. GDF11 interacts with TGF-ß receptor, resulting in activation of the ERK/EIF4E pathway, which enhances the expression of proteins for angiogenesis and anti-apoptosis, and augment of EC markers during cultured with VEGFA. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01 and ***P < 0.001. BCL-2 for B-cell lymphoma-2 protein; BAX for Bcl2 Associated X Protein