Maternal immune activation leads to defective brain blood vessels and intracerebral hemorrhages in male offspring (2022)
Figure 2

Figure 2. Maternal immune activation causes in sex-specific alterations of the expression of vascular and inflammatory controller molecules.

A. qPCR analyses of GD9+6h male embryos for: Pecam1 (Ctrl = 1.00 ± 0.17 N=8, Poly I:C = 0.89 ± 0.23 N=13), CD248 (Ctrl = 1.00 ± 0.20 N=6, Poly I:C = 1.35 ± 0.31 N=11), Vegfa (Ctrl = 1.00 ± 0.19 N=8, Poly I:C = 0.15 ± 0.04 N=8, Student's t test, ***p < .001), Pdfgb (Ctrl = 1.00 ± 0.23 N=5, Poly I:C = 1.10 ± 0.18 N=13), Tgfb1 (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.76 ± 0.22 N=10, Mann-Whitney U test, *p < .05), Tgfb2 (Ctrl = 1.00 ± 0.28 N=6, Poly I:C = 1.37 ± 0.28 N=13), Tgfb3 (Ctrl = 1.00 ± 0.51 N=6, Poly I:C = 0.73 ± 0.19 N=10), Foxf2 (Ctrl = 1.00 ± 0.45 N=8, Poly I:C = 0.46 ± 0.18 N=13) mRNAs.

B. qPCR analyses of E17 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.63 N=10, Poly I:C = 3.07 ± 1.58 N=8; Mann-Whitney U test, *p < .05), CD248 (Ctrl = 1.00 ± 0.23 N=10, Poly I:C = 3.33 ± 0.79 N=9; Mann-Whitney U test, **p < .01), Vegfa (Ctrl = 1.00 ± 0.10 N=8, Poly I:C = 1.70 ± 0.29 N=5; Mann-Whitney U test, *p < .05), Pdfgb (Ctrl = 1.00 ± 0.75 N=9, Poly I:C = 2.05 ± 0.96 N=9; Mann-Whitney U test, *p < .05), Tgfb1 (Ctrl = 1.00 ± 0.16 N=7, Poly I:C = 15.27 ± 5.75 N=7; Mann-Whitney U test, ***p < .001), Tgfb2 (Ctrl = 1.00 ± 0.16 N=8, Poly I:C = 3.82 ± 1.05 N=6; Mann-Whitney U test, *p < .05), Tgfb3 (Ctrl = 1.00 ± 0.10 N=9, Poly I:C = 10.58 ± 6.73 N=8; Mann-Whitney U test, ***p < .001); Foxf2 (Ctrl = 1.00 ± 0.06 N=5, Poly I:C = 2.60 ± 0.57 N=8; Mann-Whitney U test, *p < .05) mRNAs.

C. qPCR analyses of P90 male cortices for: Pecam1 (Ctrl = 1.00 ± 0.18 N=8, Poly I:C = 0.85 ± 0.08 N=8), CD248 (Ctrl = 1.00 ± 0.20 N=9, Poly I:C = 0.93 ± 0.11 N=8), Vegfa (Ctrl = 1.00 ± 0.07 N=8, Poly I:C = 0.75 ± 0.06 N=8; Mann-Whitney U test, *p < .05); Pdfgb (Ctrl = 1.00 ± 0.20 N=8, Poly I:C = 1.11 ± 0.17 N=4), Tgfb1 (Ctrl = 1.00 ± 0.29 N=5, Poly I:C = 2.37 ± 0.47 N=7; Mann-Whitney U test, *p < .05); Tgfb2 (Ctrl = 1.00 ± 0.29 N=4, Poly I:C = 1.19 ± 0.26 N=7), Tgfb3 (Ctrl = 1.00 ± 0.18 N=10, Poly I:C = 1.78 ± 0.50 N=11) mRNAs.

D. qPCR analyses of GD9+6h female embryos for: Pecam1 (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.22 ± 0.50 N=6), CD248 (Ctrl = 1.00 ± 0.27 N=10, Poly I:C = 2.32 ± 0.57 N=6; Mann-Whitney U test, *p < .05), Vegfa (Ctrl = 1.00 ± 0.38 N=8, Poly I:C = 0.29 ± 0.03 N=5; Mann-Whitney U test, *p < .05), Pdfgb (Ctrl = 1.00 ± 0.36 N=10, Poly I:C = 1.31 ± 0.47 N=6), Tgfb1 (Ctrl = 1.00 ± 0.16 N=8, Poly I:C = 1.63 ± 0.42 N=7), Tgfb2 (Ctrl = 1.00 ± 0.33 N=9, Poly I:C = 1.75 ± 0.29 N=6; Mann-Whitney U test, *p < .05), Tgfb3 (Ctrl = 1.00 ± 0.29 N=9, Poly I:C = 1.33 ± 0.51 N=6), Foxf2 (Ctrl = 1.00 ± 0.28 N=9, Poly I:C = 1.71 ± 0.52 N=6) mRNAs.

E. qPCR analyses of E17 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.34 N=6, Poly I:C = 0.27 ± 0.08 N=8), CD248 (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.04 ± 0.28 N=9), Vegfa (Ctrl = 1.00 ± 0.70 N=5, Poly I:C = 0.32 ± 0.13 N=5), Pdfgb (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 0.19 ± 0.07 N=9), Tgfb1 (Ctrl = 1.00 ± 0.33 N=5, Poly I:C = 0.09 ± 0.03 N=6), Tgfb2 (Ctrl = 1.00 ± 0.32 N=7, Poly I:C = 0.45 ± 0.11 N=8), Tgfb3 (Ctrl = 1.00 ± 0.38 N=7, Poly I:C = 0.40 ± 0.20 N=9), Foxf2 (Ctrl = 1.00 ± 0.18 N=5, Poly I:C = 1.04 ± 0.28 N=8) mRNAs.

F. qPCR analyses of P90 female cortices for: Pecam1 (Ctrl = 1.00 ± 0.15 N=5, Poly I:C = 0.69 ± 0.15 N=5), CD248 (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.58 ± 0.09 N=5), Vegfa (Ctrl = 1.00 ± 0.38 N=6, Poly I:C = 1.53 ± 0.38 N=8), Pdfgb (Ctrl = 1.00 ± 0.16 N=9, Poly I:C = 1.77 ± 0.35 N=17), Tgfb1 (Ctrl = 1.00 ± 0.26 N=13, Poly I:C = 1.30 ± 0.23 N=14), Tgfb2 (Ctrl = 1.00 ± 0.27 N=11, Poly I:C = 0.59 ± 0.06 N=4), Tgfb3 (Ctrl = 1.00 ± 0.45 N=11, Poly I:C = 0.51 ± 0.06 N=4) mRNAs.

G. qPCR analyses of GD9+6h male embryos for Il1α (Ctrl = 1.00 ± 0.29 N=7, Poly I:C = 2.10 ± 0.57 N=17), Il1β (Ctrl = 1.00 ± 0.39 N=6, Poly I:C = 10.84 ± 4.70 N=6; Mann-Whitney U test, **p < .01), Il1rn (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 3.60 ± 1.58 N=11), Il6 (Ctrl = 1.00 ± 0.17 N=6, Poly I:C = 0.82 ± 0.12 N=7) mRNAs.

H. qPCR analyses of E17 male cortices for: Il1α (Ctrl = 1.00 ± 0.27 N=9, Poly I:C = 1.77 ± 0.53 N=11), Il1β (Ctrl = 1.00 ± 0.21 N=7, Poly I:C = 1.33 ± 0.33 N=8), Il1rn (Ctrl = 1.00 ± 0.25 N=10, Poly I:C = 0.82 ± 0.40 N=18), Il6 (Ctrl = 1.00 ± 0.24 N=7, Poly I:C = 2.17 ± 0.43 N=8) mRNAs.

I. qPCR analyses of P90 male cortices for: Il1α (Ctrl = 1.00 ± 0.24 N=8, Poly I:C = 1.51 ± 0.26 N=10), Il1β (Ctrl = 1.00 ± 0.27 N=8, Poly I:C = 1.19 ± 0.34 N=8), Il1rn (Ctrl = 1.00 ± 0.31 N=18, Poly I:C = 1.30 ± 0.50 N=14), Il6 (Ctrl = 1.00 ± 0.27 N=7, Poly I:C = 2.40 ± 0.70 N=8) mRNAs.

J. qPCR analyses of GD9+6h female embryos for: Il1α (Ctrl = 1.00 ± 0.93 N=6, Poly I:C = 0.03 ± 0.00 N=4), Il1β (Ctrl = 1.00 ± 0.61 N=5, Poly I:C = 0.17 ± 0.04 N=7), Il1rn (Ctrl = 1.00 ± 0.25 N=9, Poly I:C = 0.33 ± 0.11 N=7), Il6 (Ctrl = 1.00 ± 0.48 N=7, Poly I:C = 0.59 ± 0.24 N=5) mRNAs.

K. qPCR analyses of E17 female cortices for: Il1α (Ctrl = 1.00 ± 0.31 N=5, Poly I:C = 1.38 ± 0.20 N=12), Il1β (Ctrl = 1.00 ± 0.30 N=5, Poly I:C = 1.05 ± 0.35 N=7), Il1rn (Ctrl = 1.00 ± 0.55 N=6, Poly I:C = 0.45 ± 0.22 N=6), Il6 (Ctrl = 1.00 ± 0.26 N=7, Poly I:C = 0.94 ± 0.14 N=8) mRNAs.

L. qPCR analyses of P90 female cortices for Il1α (Ctrl = 1.00 ± 0.08 N=5, Poly I:C = 1.32 ± 0.11 N=9), Il1β (Ctrl = 1.00 ± 0.13 N=5, Poly I:C = 0.65 ± 0.09 N=8), Il1rn (Ctrl = 1.00 ± 0.61 N=4, Poly I:C = 0.85 ± 0.29 N=7), Il6 (Ctrl = 1.00 ± 0.11 N=9, Poly I:C = 1.28 ± 0.20 N=20) mRNAs. Values are normalized over control (dashed line).

M. ELISA quantification of total TGF-β1 in E17 male embryos brains as pg/mL (Ctrl = 132.6 ± 18.14, Poly I:C = 129.6 ± 8.62).

N. ELISA quantification of active free TGF-β1 in E17 male embryos brains as pg/mL (Ctrl = undetectable, Poly I:C = 0.92 ± 0.25).

O. ELISA quantification of total TGF-β1 in E17 female embryos brains as pg/mL (Ctrl = 177.6 ± 10.88, Poly I:C = 134.4 ± 13.59).

P. ELISA quantification of active free TGF-β1 in E17 female embryos brains as pg/mL (undetectable).

Q-S. Western Blotting quantification of SMAD proteins in E17 male cortices for (Q) pSMAD1-5 (Ctrl = 0.99 ± 0.21, Poly I:C = 2.67 ± 0.46); Mann-Whitney U test, *p < .05, (R) pSMAD2 (Ctrl = 1.00 ± 0.04, Poly I:C = 0.88 ± 0.24), (S) SMAD4 (Ctrl = 1.00 ± 0.12, Poly I:C = 0.96 ± 0.21). Protein levels were normalized to GAPDH.

T-V. Western Blotting quantification of SMAD proteins in E17 female cortices for (T) pSMAD1-5 (Ctrl = 1.00 ± 0.02, Poly I:C = 0.70 ± 0.18), (U) pSMAD2 (Ctrl = 1.00 ± 0.01, Poly I:C = 1.09 ± 0.12), (V) SMAD4(Ctrl = 1.00 ± 0.16, Poly I:C = 1.46 ± 0.57. Protein levels were normalized to GAPDH.

Data information: Ctrl males = white bars with blu border. Poly I:C males = blu bars with blu border. Ctrl females = white bars with purple border. Poly I:C females = purple bars with purple border. Numbers in bars indicate the number of animals (N). Bars represent mean ± SEM.