Table 1.
Primers for quantitative real-time PCR.
Fig 1.
Increase in decidual VEGF expression is associated with increase in macrophage number and dramatic upregulation of M2 markers in the decidua.
A-D, representative photomicrographs (in situ hybridization; A-B, dark-field images; C-D, bright-field images) show a marked increase in VEGF mRNA expression in first trimester decidua (B, D) as compared with secretory phase endometrium (A, C). Immunofluorescence (E-F, CD68 immunostaining; G-H, DAPI staining) show a concomitant increase in CD68-positive macrophages in first trimester decidua (F) as compared to secretory phase endometrium (E). Scale bars: A-D, 100 μm and E-H, 40 μm. I, qPCR analysis of VEGF and M1 (CXCL10, CCR7, and IL12) and M2 (CD206, CD163, and CCL17) markers in first trimester decidual tissue compared with secretory phase endometrium. J-K, qPCR analysis of M1 (J) and M2 (K) markers in decidual samples from preeclamptic pregnancies compared with gestational-age matched controls. Note the significant increase in M1 markers and decrease in M2 markers in decidual samples from preeclamptic pregnancies. Graphs represent means ± standard errors; * = p ≤0.05, ** = p <0.01, and *** = p <0.001.
Fig 2.
THP1 cells differentiated into polarized macrophages display characteristic morphology and markers of polarization.
A-D, photomicrographs showing characteristic morphologies of THP1 cells (A), and differentiated M0 (B), M1 (C), and M2 (D) macrophages. M1 macrophages (C) appear flattened as compared with M0 cells (B), and M2 macrophages take on a more elongated shape (D). E-F, qPCR quantification of M1 (E) and M2 (F) macrophage markers in THP1-derived M1 and M2 cells, respectively, relative to M0 cells. The expression of M1 and M2 mRNA markers is consistent with the observed morphologies of the M1 (C) and M2 (D) macrophages. Graphs represent means ± standard error. Scale bar = 30 μm; * = p < 0.05, ** = p <0.01, and **** = p <0.0001.
Fig 3.
VEGF treatment suppresses M1 markers and induces M2 markers in polarized macrophages.
A, qPCR analysis of M0, M1, and M2 cells shows that all three express both VEGF receptors 1 (VEGFR1) and 2 (VEGFR2). Note the highly significant increase in expression of VEGFR1 in M1 cells. B, initial dose-response study showing the effect of increasing concentrations of rhVEGF (0.1–20 μg/mL). VEGF induces an upregulation of M2 markers and downregulation of M1 markers in THP1-derived M1 cells, with highest response seen at 4 μg/mL. C-D, VEGF treatment at 4 μg/mL caused an increase in M2 markers and a decrease in M1 markers in both THP1-derived M1 (C) and M2 (D) macrophages. Graphs represent means ± standard errors; * = p < 0.05, ** = p <0.01.
Fig 4.
Decidual endometrial stromal cells produce VEGF and stimulate M2 macrophage polarization.
A, qPCR analysis of VEGF mRNA expression in decidualized and non-decidualized stromal cells. VEGF expression is highly significantly increased following decidualization. B, expression of M1 and M2 mRNA markers following treatment with conditioned medium from decidualized/non-decidualized stromal cells, with or without the VEGF inhibitor sFlt1. Decidualized stromal cell conditioned medium induces a significant decrease in all M1 markers and a significant increase in all M2 markers, but the effect of the conditioned medium on M2 markers was abrogated by sFlt1 treatment, suggesting a potential role for VEGF in M2 polarization. Graphs represent means ±standard errors; * = p < 0.05 compared to controls; # = p<0.05 compared to decidualization medium; **** = p <0.0001.
Fig 5.
VEGF produced by decidualized endometrial stromal cells stimulate macrophage migration.
A-C, transwell migration assay showing migration of M0 cells without (A) or with (B) VEGF treatment (2 μg/mL). Bar graph (C) shows quantitative estimation of the number of migrated cells. Addition of VEGF to the medium in the lower chambers induced about a 3-fold increase in migration of cells (B and C). D-G, transwell migration assay of M0 cells; D, treatment with non-decidualized stromal cell-conditioned medium; E-F, treatment with decidualized stromal cell-conditioned medium in the absence (E) or presence (F) of the VEGF inhibitor sFlt1; G, quantification of migrated cells. Note that decidualized stromal cell-conditioned medium significantly stimulated migration of M0 cells (E and G) but the effect was completely abrogated by the addition of sFlt1 (F), suggesting that VEGF secreted into the conditioned medium may play a role in stimulating the migration of these cells. Graphs represent means ± standard errors; * = p<0.05, ** = p<0.01, *** = p<0.001. Scale bars represent 50 μm.
Fig 6.
Model of VEGF action on decidual macrophages.
Decidual stromal cells produce VEGF, which acts to recruit monocytes and tissue macrophages to the site of implantation in normal pregnancy. It also acts to polarize macrophages to the M2 phenotype. In preeclampsia, however, there is an increase in VEGF, which may enhance recruitment of macrophages to the decidua. Excess VEGF at the implantation site in preeclamptic pregnancies may also stimulate excess production of sFlt1 by trophoblast cells (22, 23), leading to complete inhibition of VEGF signaling (lack of free VEGF; increase in VEGF-sFlt1 bound form), and ultimately resulting in the failure of differentiation of macrophages to the M2 phenotype and accumulation of large numbers of M1 cells in the placental bed.