Fig 2: Angiopoietin‐like 4 is expressed in spheroid cultures of fibroblasts of the periodontal ligament. Human fibroblasts of the periodontal ligament (PDLF) in spheroid cultures were incubated in hypoxia or stimulated with L‐mimosine (L‐MIM) in medium with serum. Angiopoietin‐like 4 was measured at mRNA level (Angptl4; A) and protein level (ANGPTL4; B) using qPCR and ELISA, respectively. Bars represent mean + standard deviation, relative to the normoxic control. Experiments were performed three times with two different donors, respectively (N = 6). *P < 0.05 vs control (dashed line; A, white bar; B)

Fig 3: Angiopoietin‐like 4 in monolayer cultures of fibroblasts of the periodontal ligament stimulated with P. gingivalis lipopolysaccharide, E. coli lipopolysaccharide, IL‐1β, and TNFα in the presence of hypoxia or L‐mimosine. Human fibroblasts of the periodontal ligament (PDLF) in monolayer cultures were treated with P. gingivalis lipopolysaccharide (LPS Pg, A(i), B(i)), E. coli lipopolysaccharide (LPS Ec, A(ii), B(ii)), IL‐1β, A(iii), B(iii), TNFα, A(iv), B(iv), with and without the presence of hypoxia or L‐mimosine in medium without serum. Angiopoietin‐like 4 was measured at mRNA levels (Angptl4; A) and protein levels (ANGPTL4; B) using qPCR and ELISA, respectively. Bars represent mean + standard deviation, relative to the normoxic control. Experiments were performed twice with three different donors, respectively (N = 6). *P < 0.05 vs control (dashed line; A, white bar; B)

Fig 4: IMRCs significantly promote endometrial angiogenesis in IUA patients(A–C) The hysteroscopic image (A), H&E staining (B), and Masson staining (C) of the endometrium of patient 16 at V3 after cell injection. Scale bar, 100 µm. The hysteroscopic image (A) of patient 16 at visit V3 is also presented in Figure S5A, as both figures correspond to the same patient at the same follow-up time point. (D and E) The representative images (D) and quantitative analysis (E) of immunofluorescence of CD31 and α-SMA in the normal proliferative endometrium, IUA patient endometrium, and the endometrium of patient 16 at V3. Scale bar, 50 μm. (F and G) The representative images (F) and quantitative analysis (G) of immunohistochemistry of ANGPTL4 in normal proliferative endometrium, endometrium of patient 16 at V0, and endometrium of patient 16 at V3. Scale bar, 100 µm. The dots in the bar chart represent the number of biological replicates, which is three. ∗p < 0.05; ∗∗p < 0.01. Data shown as mean ± SD.

Fig 5: ANGPTL4 plays a key role in co-culture-CM-induced angiogenesis(A) The protein concentration of ANGPTL4 was measured by ELISA among ESC-CM, IMRC-CM, and co-culture-CM groups, respectively. (B and C) The mRNA expression of ANGPTL4 in ESCs (B) and IMRCs (C) was measured by quantitative real-time PCR. Co-cultured ESCs: ESC after co-culturing with IMRC; co-cultured IMRCs: IMRCs after co-cultured with ESCs. (D) The protein expression of ANGPTL4 in co-cultured ESCs was measured by Western blotting. (E) The relative expression of ANGPTL4 after siRNA-mediated knockdown of ANGPTL4 in ESCs. (F–H) Co-culture-CM-induced angiogenesis was impaired after the knockdown of ANGPTL4 in ESCs. Ctrl, control, basal medium for IMRCs; si-NC, si-ANGPTL4, siRNA of ANGPTL4; siRNA of negative control. Scale bar, 100 μm. (I–K) Angiogenesis increased in the rHuANGPTL4 group compared to that in the Ctrl group. A similar result was observed in the co-culture-CM and rHuANGPTL4 combined groups in comparison with that in co-culture-CM alone. The concentration of rHuANGPTL4 was 1 μg/mL. Scale bar, 100 μm. The dots in the bar chart represent the number of technical replicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. Data shown as mean ± SD.