Impaired Angiogenic Function of Fetal Endothelial Progenitor Cells via PCDH10 in Gestational Diabetes Mellitus

Abstract

Maternal hyperglycemia, induced by gestational diabetes mellitus (GDM), has detrimental effects on fetal vascular development, ultimately increasing the risk of cardiovascular diseases in offspring. The potential underlying mechanisms through which these complications occur are due to functional impairment and epigenetic changes in fetal endothelial progenitor cells (EPCs), which remain less defined. We confirm that intrauterine hyperglycemia leads to the impaired angiogenic function of fetal EPCs, as observed through functional assays of outgrowth endothelial cells (OECs) derived from fetal EPCs of GDM pregnancies (GDM-EPCs). Notably, PCDH10 expression is increased in OECs derived from GDM-EPCs, which is associated with the inhibition of angiogenic function in fetal EPCs. Additionally, increased PCDH10 expression is correlated with the hypomethylation of the PCDH10 promoter. Our findings demonstrate that in utero exposure to GDM can induce angiogenic dysfunction in fetal EPCs through altered gene expression and epigenetic changes, consequently increasing the susceptibility to cardiovascular diseases in the offspring of GDM mothers.

Keywords: PCDH10; angiogenesis; endothelial progenitor cells; epigenetic changes; gestational diabetes mellitus.

Figure A1

Schematic image of the differentiation of EPCs to OECs.

Figure A2

Lentiviral transfection workflow for PCDH10 knockdown as described in the Section 4.

Figure 1

GDM impaired the endothelial angiogenic capacity in fetal EPCs. (A,B) Cell migration assay using a Transwell chamber after culture for 4 h, showing representative images of the H&E staining (A) and quantification (B). (C,D) Cell–matrix adhesion assay after incubation for 30 min, showing representative images of H&E staining (C) and quantification (D). (E,F) Tube formation assay following cell incubation on Matrigel for 18–24 h, showing representative images (E) and quantification (F). (G) Proliferation assay using MTT; the absorbance value (OD) of each well was measured at 490 nm at 24 h, 48 h, and 72 h. All data are presented as the mean ± SE. ** p < 0.01. n = 3 independent experiments for each assay.

Figure 2

Exposure to high-glucose conditions reduced the angiogenic capacity in fetal EPCs, similar to that in cells from GDM. N-EPCs were exposed to normal D-glucose (5 mM) or high D-glucose (30 mM) concentrations. (A,B) Cell migration assay using a Transwell chamber after culture for 4 h, showing representative images of H&E staining (A) and quantification (B). (C,D) Cell–matrix adhesion assay after incubation for 30 min, showing representative images of H&E staining (C) and quantification (D). (E,F) Tube formation assay following cell incubation on Matrigel for 18–24 h, showing representative images (E) and quantification (F). (G) Proliferation assay using MTT; the absorbance value (OD) of each well was measured at 490 nm at 72 h. All data are presented as the mean ± SE. ** p < 0.01. n = 3 independent experiments for each assay.

Figure 3

Gene expression patterns of OECs from GDM-EPCs compared with those from N-EPCs. (A) Pie charts show the transcripts significantly up-regulated and down-regulated in OECs from GDM-EPCs relative to the normal pregnancy. (B) Gene ontology analyses included genes that showed a p-value < 0.05 and a fold change > 2.0 and were performed using DAVID Bioinformatics Resources.

Figure 4

Significantly increased PCDH10 expression in OECs from GDM-EPCs and in OECs from N-EPCs exposed to high-glucose conditions. (A) PCDH10 expression using qRT-PCR in OECs derived from N-EPCs and GDM-EPCs. (B) PCDH10 expression using qRT-PCR in OECs from N-EPCs exposed to normal glucose (5 mM) and high-glucose conditions (30 mM). Data are shown as the mean ± SE from three independent experiments. ** p < 0.01. NL, normal pregnancy; GDM, gestational diabetes mellitus; NG, normal glucose.

Figure 5

Knockdown of PCDH10 expression in OECs from GDM-EPCs reversed the suppression of angiogenesis. (A) Knockdown efficiency using qRT-PCR in OECs derived from GDM-EPCs after transfection with shPCDH10. (B) Representative image of Western blots of shPCDH10-transfected OECs from GDM-EPCs. (C,D) Cell migration assay using a Transwell chamber after culture for 4 h, showing representative images of H&E staining (C) and quantification (D). (E,F) Cell–matrix adhesion assay after incubation for 30 min, showing representative images of H&E staining (E) and quantification (F). (G,H) Tube formation assay following cell incubation on Matrigel for 18–24 h, showing representative images (G) and quantification (H). (I) Proliferation assay using MTT; the absorbance value (OD) of each well was measured at 490 nm at 72 h. All data are presented as the mean ± SE. ** p < 0.01, comparison was performed between normal and GDM groups, and between the shCtrl- and shPCDH10-transfected groups.

Figure 6

Exposure to high-glucose conditions increased PCDH10 expression in OECs from N-EPCs, and the increased expression of PCDH10 was not reversed under normal glucose conditions. (A) Schematic diagram of the procedure to evaluate the association of PCDH 10 expression and glucose conditions. (B) The expression of PCDH10 in OECs under high-glucose conditions for 19 d followed by normal glucose conditions. High-glucose-induced increased PCDH10 expression occurred even in the early passages of OECs, and the changes were not reversed even after return to normal conditions. (C–E) The expression of PCDH10 in OECs derived from N-EPCs exposed to high-glucose conditions for 10 d, 19 d, and 59 d. ** p < 0.01. NG, normal glucose; HG, high glucose.

Figure 7

The promoter CGIs of PCDH10 were hypomethylated in OECs from GDM-EPCs and N-EPCs exposed to high-glucose conditions, and correlated with increased PCDH10 expression. (A) Schematic diagram of the CpG island of the PCDH10 promoter. (B) Methylation status of PCDH10 in OECs derived from GDM. (C) Relative mRNA expression of PCDH10 in OECs derived from GDM. (D) Methylation status of PCDH10 in OECs derived from N-EPCs exposed to normal and high-glucose conditions. (E) Relative mRNA expression of PCDH10 in OECs derived from N-EPCs under normal glucose and high-glucose conditions. (F,G) Treatment with 5-Aza-dC induced hypomethylation of the PCDH10 promoter CGIs with concomitant increased PCDH10 mRNA expression in a dose-dependent manner. ** p < 0.01. NL, normal pregnancy; GDM, gestational diabetes mellitus; NG, normal glucose.