Dysregulation of integrin αvβ3 and α5β1 impedes migration of placental endothelial cells in fetal growth restriction

Abstract

Placentas from pregnancies complicated by severe early-onset fetal growth restriction (FGR) exhibit diminished vascular development mediated by impaired angiogenesis, but underlying mechanisms remain unknown. In this study, we show that FGR endothelial cells demonstrate inherently reduced migratory capacity despite the presence of fibronectin, a matrix protein abundant in placental stroma that displays abnormal organization in FGR placentas. Thus, we hypothesized that aberrant endothelial-fibronectin interactions in FGR are a key mechanism underlying impaired FGR endothelial migration. Using human fetoplacental endothelial cells isolated from uncomplicated term control and FGR pregnancies, we assessed integrin α5β1 and αvβ3 regulation during cell migration. We show that endothelial integrin α5β1 and αvβ3 interactions with fibronectin are required for migration and that FGR endothelial cells responded differentially to integrin inhibition, indicating integrin dysregulation in FGR. Whole-cell expression was not different between groups. However, there were significantly more integrins in focal adhesions and reduced intracellular trafficking in FGR. These newly identified changes in FGR endothelial cellular processes represent previously unidentified mechanisms contributing to persistent angiogenic deficiencies in FGR.

Keywords: Angiogenesis; Endothelial cells; Fetal growth restriction; Human; Integrins; Placental dysfunction.

Fig. 1.

FGR endothelial cell migratory impairment persists in the setting of exogenous fibronectin substrate. (A) Representative images of percent relative wound density (%RWD) with automated image analyses over 24 h for control and FGR endothelial cells plated on fibronectin. (B) Comparison of migratory capacity of control and FGR subjects plated on fibronectin showed a significant reduction in FGR endothelial cell migration compared with control cells (P<0.0001; non-linear regression). Subject number of n=6 per group. Data are mean±s.e.m. Experiments were repeated in triplicate, with at least four technical replicates per subject per condition.

Fig. 2.

Integrin αvβ3 is ineffectively used by FGR endothelial cells. (A,B) Endothelial cell surface inhibition of either active integrin α5 (JBS5; B) or active integrin β1 (AIIB2; A) resulted in significantly reduced migration in a scratch wound assay for both control and FGR subjects. (C) Although inhibition of integrin αvβ3 (LM609) significantly reduced control endothelial cell migration to the degree of basal impairment seen in FGR cells, there was no additional effect of αvβ3 blockade on FGR cell migration. All P-values are listed in the corresponding tables. Subject number of n=6 per group per treatment condition. Data are mean±s.e.m. Non-linear regression was used for statistical comparisons. Experiments were repeated in triplicate, with at least four technical replicates per subject per condition.

Fig. 3.

FGR migratory impairment on fibronectin is not caused by reduced expression of integrins αvβ3 and α5β1. (A) There was no significant difference in mRNA expression of integrins α5, β1, αv and β3 between control and FGR endothelial cells. (B) Via capillary immunoblotting, protein expression of these integrin subunits also did not significantly differ between control and FGR endothelial cells after normalization to total protein stain (shown in blue). Subject number of n=6 per group. Unpaired two-tailed Student's t-tests were used for statistical comparisons. Horizontal line shows mean. Experiments were repeated in triplicate.

Fig. 4.

FGR and control endothelial cells display similar amounts of active integrin α5β1 and αvβ3 at the cell membrane. (A-D) After isolation of integrin-based adhesions and complexes at the cell membrane, samples were probed for integrin α5 (A), integrin αv (B), integrin β3 (C) and integrin β1 (D). Relative expression was quantified after normalization to total protein (shown in blue). Subject number of n=6 per group with six plates pooled per subject. Mann–Whitney U-tests were used for statistical analysis. Data are mean±s.e.m. Integrin β1 was not analyzed statistically as there was no signal present at the expected molecular weight.

Fig. 5.

Integrin α5β1 and αvβ3 focal adhesion complexes are altered in FGR endothelial cells. (A) Details of the experimental design using TIRF imaging followed by the Focal Adhesion Analysis Server (FAAS) for quantification. (B-F) Representative raw images and masks demonstrate significantly increased numbers of integrin αvβ3-paxillin (B; eight ROIs per subject, P=0.0096) and αvβ3-vinculin (C; eight ROIs per subject, P=0.0039). There were also significantly more numbers of active integrin a5 (D; 32 ROIs per subject, P=0.0276), α5-zyxin (E; eight ROIs per subject, P=0.0281) and β1-vinculin (F; eight ROIs per subject, P=0.0027). Three subjects (two control and one FGR) were removed from analysis due to positive fluorescent signal with secondary antibody only staining, with n=4 controls and n=5 FGR subjects. Green horizontal lines represent the mean for normally distributed data or median for non-normally distributed data. Unpaired two-tailed Student's t-tests were used for statistical comparisons when data were parametric (C-F), and Mann–Whitney U-tests were used for non-parametric data (B). Scale bars: 20 μm.

Fig. 6.

Intracellular endosomal vesicles are reduced in FGR endothelial cells. (A) A graphical depiction of integrin intracellular trafficking during cell locomotion. (B,C) Representative images and graphical quantification demonstrate a significant reduction in early endosomes (B; EEA1, P<0.0001) and late endosomes (C; Rab7, P=0.0026) in FGR endothelial cells. (D-F) In contrast, representative images and quantification show similar amounts of internalized active integrin α5 (D), active integrin β1 (E) and active integrin αvβ3 (F) between control and FGR endothelial cells. n=5 controls and n=6 FGR subjects. One control subject was removed from analysis due to a high intensity secondary antibody threshold, which limited validation of the primary stain. At least 72 images containing 5-10 cells per image were analyzed per subject per stain. Green horizontal lines represent the mean for normally distributed data or median for non-normally distributed data. Unpaired two-tailed Student's t-tests were used for statistical comparisons when data were parametric (B,F), and Mann–Whitney U-tests were used for non-parametric data (C-E). Scale bars: 50 μm.