Fig 1: HHcy inhibits VEGFA expression and secretion by upregulating CD36 expression in DSCs, thereby impairing angiogenesis. A The VEGFA mRNA expression of DSCs after treatment with Hcy and/or 100µM SSO (n = 3 per group). B and C The VEGFA protein expression of DSCs after treatment with Hcy and/or 100µM SSO (n = 3 per group). D VEGFA concentration in the culture supernatant of DSCs after treatment with Hcy and/or 100µM SSO, measured by ELISA (n = 6 per group). E–G Conditioned medium (CM) collected from treated DSCs was used for HUVECs tube formation assays. Images were taken 6 h after incubation. Scale bar = 100 μm. Quantitative analysis of branch number and total tube length (n = 3 per group). H CM from treated DSCs was used for HUVECs wound healing assays. Images were taken at 0 h and 12 h. Scale bar = 50 μm. The wound healing rate was quantified (n = 3 per group). The data were expressed as mean ± SEM and statistical comparisons were assessed by unpaired two-tailed Student’s t-test. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001
Fig 2: Schematic diagram of this study. HHcy leads to lipid deposition in decidua by upregulating CD36 expression in DSCs, which suppresses VEGFA expression and secretion possibly by activating the PPAR signaling pathway, thereby impairing angiogenesis at the maternal-fetal interface and causing FGR. The diagram was created at https://BioRender.com
Fig 3: HHcy impairs vascular density at the maternal-fetal interface and affects angiogenic factor expression in decidua. A Representative images and quantification of CD31 immunofluorescence staining in decidua of ND and HMD pregnant rats at E10 (n = 4 per group). Scale bar = 200 μm. B Representative images and expression of CD31 immunofluorescence staining in placentas at E14 (n = 4 per group). Scale bar = 100 μm. C Representative images and expression of CD31 immunofluorescence staining in placentas at E20 (n = 4 per group). Scale bar = 100 μm. D Protein expression levels of VEGFA, PLGF, HIF1α, and CA9 in decidua at E10 (n = 6 per group). The data were expressed as mean ± SEM and statistical comparisons were assessed by unpaired two-tailed Student’s t-test. ns P > 0.05, * P < 0.05, ** P < 0.01
Fig 4: HHcy inhibits angiogenesis by reducing VEGFA secretion from DSCs. A Cell viability of DSCs assessed by CCK8 assay after treatment with different concentrations of Hcy for 24 h (n = 6 per group). B VEGFA mRNA expression in DSCs following treatment with varying concentrations of Hcy for 24 h (n = 4 per group). C and D VEGFA protein levels in DSCs treated with normal culture medium (NC) or medium containing 4000 µM Hcy (HHcy) for 24 h (n = 4 per group). E VEGFA mRNA levels in NC and HHcy groups (n = 4 per group). F VEGFA concentration in DSCs culture supernatants, analyzed by ELISA (n = 6 per group). G–I CM from DSCs was used for HUVECs tube formation assays. Images were taken 6 h post-seeding. Scale bar = 100 μm. Quantitative analysis of tube branch number and total tube length (n = 3 per group). J and K CM from DSCs was used for HUVECs wound healing assays. Scratch images were taken at 0 and 12 h. Scale bar = 50 μm. Wound healing rates were quantified (n = 3 per group). The data were expressed as mean ± SEM and statistical comparisons were assessed by unpaired two-tailed Student’s t-test. ns P > 0.05, * P < 0.05, *** P < 0.001, **** P < 0.0001
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