PlGF knockout delays brain vessel growth and maturation upon systemic hypoxic challenge

Abstract

In this study, we have investigated the potential role of placental growth factor (PlGF) in hypoxia-induced brain angiogenesis. To this end, PlGF wild-type (PlGF(+/+)) and PlGF knockout (PlGF(-/-)) mice were exposed to whole body hypoxia (10% oxygen) for 7, 14, and 21 days. PlGF(+/+) animals exhibited a significant ~40% increase in angiogenesis after 7 days of hypoxia compared with controls, while in PlGF(-/-) this effect only occurred after 14 days of hypoxia. No differences in pericyte/smooth muscle cell (SMC) coverage between the two genotypes were observed. After 14 days of hypoxia, PlGF(-/-) microvessels had a significant increase in fibrinogen accumulation and extravasation compared with those of PlGF(+/+), which correlated with endothelial cell disruption of the tight junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked both pericyte/SMC coverage and endothelial vascular endothelial growth factor expression, and regressed after 21 days of hypoxia. Vascular endothelial growth factor expression levels were found to be significantly lower in the frontal cortex of PlGF(-/-) compared with those in PlGF(+/+) animals during the first 5 days of hypoxia, which in combination with the lack of PlGF may have contributed to the delayed angiogenic response and the prothrombotic phenotype observed in the PlGF(-/-)animals.

Figure 1

Effects of 7-, 14-, and 21-day exposure to 10% hypoxia on body weight (A) and hematocrit (B) of PlGF^+/+ and PlGF^−/− mice. Data presented are mean values±s.e.m. of four mice per genotype and time point. ^***indicates significant difference (P<0.001) between hypoxic groups and the corresponding normoxic controls (analysis of variance (ANOVA) followed by Bonferroni post-test). PlGF, placental growth factor.

Figure 2

(A) Representative images of sections of the cerebral cortex from PlGF^+/+ and PlGF^−/− mice exposed to normoxia or 7-, 14-, or 21-day hypoxia. Sections were immunostained for the endothelial marker, CD31 (green). Scale bar=100 μm. (B) Quantification of total area of CD31⁺ cells after 7-, 14-, or 21-day normoxia or hypoxia as described in Materials and methods. (C) Number of CD31⁺ vessels separated in size intervals of 0 to 100 μm², 150 to 500 μm², and >500 μm² for each time point. White solid bars represent normoxic controls and hatched bars represent hypoxic groups. Results expressed are mean values±s.e.m. ‘^*' indicates significant difference (^*P<0.05, ^**P<0.01, ^***P<0.001) between hypoxic groups and the corresponding normoxic control groups and ‘^#' indicates significant difference (^#P<0.05; ^##P<0.01) between hypoxic PlGF^+/+ and PlGF^−/− groups (analysis of variance (ANOVA) followed by Bonferroni post-test). PlGF, placental growth factor.

Figure 3

(A) Double-immunofluorescence staining performed on sections of cerebral cortex from normoxic or hypoxic PlGF^+/+ and PlGF^−/− mice, using the endothelial marker CD31 (green) with either of the following pericyte markers: NG2 (red), desmin (red), or vascular smooth muscle cell (VSMC) marker α-smooth muscle actin (α-SMA) (red). Scale bar=100 μm. Insert in the right upper corner represents a magnified image of an NG2⁺ reactive pericyte. (B) Quantitative analysis of total area of NG2⁺, desmin⁺, and α-SMA⁺ cells as described in Materials and methods. White solid bars represent normoxic controls and hatched bars represent hypoxic groups. Results expressed are mean values±s.e.m. ‘^*' indicates significant difference (^*P<0.05, ^**P<0.01, ^***P<0.001) between hypoxic groups and the corresponding normoxic control groups and ^# indicates significant difference (P<0.05) between hypoxic PlGF^+/+ and PlGF^−/− groups (analysis of variance (ANOVA) followed by Bonferroni post-test). PlGF, placental growth factor.

Figure 4

(A, B) Double-immunofluorescence staining was performed on sections of cerebral cortex from normoxic and hypoxic PlGF^+/+ and PlGF^−/− mice, using the endothelial marker CD31 (green) and fibrinogen (red). Arrows indicate the presence of fibrinogen in the lumen of PlGF^−/− vessels after 14 days hypoxia (B). Scale bar=100 μm. (C) Quantitative analysis of total area of fibrinogen⁺ microvessels from PlGF^+/+ and PlGF^−/− mice exposed to 14- and 21-day normoxia or hypoxia. White solid bars represent normoxic controls and hatched bars represent hypoxic groups. Results expressed are mean values±s.e.m. ^* indicates significant difference (P<0.05) between hypoxic groups and the corresponding normoxic control groups, and ^## indicates significant difference (P<0.01) between hypoxic PlGF^+/+ and PlGF^−/− groups (analysis of variance (ANOVA) followed by Bonferroni post-test). (D, E) Triple-immunofluorescence for fibrinogen (red), endothelial cells (green), and astrocytes (white) on frozen sections of cerebral cortex from PlGF^−/− mice exposed to 14 days of hypoxia. Scale bar=50 μm. Arrows indicate reactive astrocytes surrounding fibrinogen⁺ vessels. (F) Double-immunofluorescence of sections of cerebral cortex from PlGF^−/− mice exposed to 14-day hypoxia, using the endothelial marker CD31 (green) and pericyte/vascular smooth muscle cell (VSMC) markers NG2, desmin, and α-smooth muscle actin (α-SMA) (red). Boxes in f1, f2, f3 outline areas magnified in f1′, f2′, f3′, showing the lack of pericyte/VSMC markers in enlarged microvessels (>500 μm² CD31⁺ vessel group). Scale bar=100 μm. (G) Triple-immunofluorescence staining of fibrinogen (red), endothelial cells (green), and vascular endothelial growth factor (VEGF) (white) in sections of cerebral cortex from PlGF^−/− mice after 14 days of hypoxia. Scale bar=50 μm. PlGF, placental growth factor.

Figure 5

Double-immunofluorescence of sections of cerebral cortex from PlGF^+/+ and PlGF^−/− mice exposed to 14-day hypoxia, using the endothelial marker CD31 (green) and tight junction marker claudin-5 (red). Arrows indicate areas of disruption of claudin-5. Scale bar=50 μm. PlGF, placental growth factor.

Figure 6

(A) Agarose gel showing placental growth factor (PlGF) and β-actin RT-PCR (reverse transcriptase polymerase chain reaction) amplification products from the frontal brain region of PlGF^+/+ and PlGF^−/− mice exposed to 5 days of normoxia (N) or 1 to 5 days of hypoxia (H). (B) Vascular endothelial growth factor (VEGF) and β-actin protein expression analyzed by Western blot from PlGF^+/+ and PlGF^−/− mice exposed to 5 days of normoxia (N) or 1 to 5 days of hypoxia (H).

Figure 7

Schematic representation of the effect of hypoxia on the brain angiogenesis in PlGF^+/+ and PlGF^−/− mice. Under normoxic conditions, PlGF^+/+ and PlGF^−/− mice exhibit similar brain vascularity. After 7 days hypoxia, an angiogenic response is induced in PlGF^+/+ mice, while in PlGF^−/− mice, this effect is delayed until 14 days of hypoxia. The delayed angiogenic response likely promotes hypoxic stress in PlGF^−/− mice, which leads to fibrinogen accumulation and a small amount of extravasation. This affects vessel integrity as indicated by the lack of endothelial vascular endothelial growth factor (VEGF) expression, pericyte coverage, and disruption of the tight junction protein claudin-5 in the fibrinogen⁺ vessels, which ultimately may result in vessel regression after 21 days of hypoxia. PlGF, placental growth factor.