Placental growth factor deficiency is associated with impaired cerebral vascular development in mice

Abstract

Study hypothesis: Placental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning.

Study finding: PGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development.

What is known already: PGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, pre-eclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgf (-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgf (-/-) mice.

Study design, samples/materials, methods: Here, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgf (-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgf (-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay.

Main results and the role of chance: Pgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain (HB). PGF and VEGF expression were similar in midbrain. Delayed HB vascularization was seen at E10.5 and 11.5 in Pgf (-/-) brains. At E14.5, Pgf (-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted post-natally. Functionally, adult Pgf (-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not.

Limitations, reasons for caution: Since Pgf (-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring.

Wider implications of the findings: These studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments.

Large scale data: N/A.

Study funding and competing interests: This work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to B.A.C. and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to R.L.L.; Queen's University to V.R.K. and the Canadian Institutes of Health Research to M.T.R. The work of P.C. is supported by the Belgian Science Policy BELSPO-IUAP7/03, Structural funding by the Flemish Government-Methusalem funding, and the Flemish Science Fund-FWO grants. There were no competing interests.

Keywords: PGF; cerebral vessels; circle of Willis; fetal development; pre-eclampsia; stroke.

Figure 1

Relative gene expression in B6 fetal brains. Quantitative PCR data are shown for relative expression of mRNA for Pgf (A), Vegfr1 (B), Vegf (C) and Vegfr2 (D) by fetal brain regions. The brains were dissected into forebrain (FB) (black bars), midbrain (MB) (gray bars) and hindbrain (HB) (white bars) at embryonic day (E) 12.5, 14.5, 16.5 and 18.5. Results were considered significant when P < 0.05 (*) or P < 0.02 (**) for comparisons between the brain areas and P < 0.05 (^#) or P < 0.02 (^##) for comparisons between the gestational times. β-actin was used as the reference gene.

Figure 2

Immunolocalization of placental growth factor (PGF) and vascular endothelial growth factor (VEGF) in embryonic day (E) 12.5 and E14.5 B6 fetal brains. Images depict immunofluorescent staining of PGF (green), VEGF (green) and CD31 (red) and nuclear staining by 4′,6-diamidino-2-phenylindole (DAPI) (blue) in fetal brain at E12.5 (A) and E14.5 (B). Two areas from each region (Fore-, mid- and hind-brain; FB, MB and HB) were chosen. The schematic drawings locate the following structures at E12.5 (telencephalic vesicle, diencephalon region, maxillary component of first branchial arch, trigeminal ganglion, vessels of marginal layer, roof of HB) and at E14.5 (superior dural venous sinus, lateral ventricle/corpus striatum, diencephalon (hypothalamus), trigeminal ganglion, transverse dural venous sinus, cells of fourth ventricle).

Figure 3

Immunolocalization of placental growth factor (PGF) and vascular endothelial growth factor (VEGF) in embryonic day (E) 16.5 and 18.5 B6 fetal brains. Images depict immunofluorescence staining of PGF (green), VEGF (green) and CD31 (red) and nuclear staining by 4′,6-diamidino-2-phenylindole DAPI (blue) in fetal brain at E16.5 (A) and E18.5 (B). Two areas from each region (Fore-, mid- and hind-brain; FB, MB and HB) were chosen. The schematic drawings show the location of the wall of telencephalon, lateral ventricles, transverse dural venous sinus, mesencephalic vesicle, primitive cerebellum, and vessels of primitive cerebellum at E16.5 as well as the vessels of white mater, superior horn of lateral ventricle, vessels of hippocampus, transverse dural venous sinus, posterior part of cerebellum, and posterior semi-circular canal at E18.5.

Figure 4

Whole mount vascular staining of hindbrain (HB) at embryonic day (E) 10.5 and 11.5. HB vascularization was compared between B6 and Pgf  ^−/− fetuses at E10.5 and 11.5 by examining the isolectin B4 (IB4) stained (white) ventricular plexus (A) and sprouting in transverse sections of the hindbrain (B). In the ventricular plexus, vessel diameter was thinner in the Pgf  ^−/− HB vasculature at E10.5 (C). Conversely, the number of junctions per area was significantly greater in the E10.5 Pgf  ^−/− HB vascular plexus (D). Confocal imaging of the HB cross-sections revealed smaller sprout diameter (E) and reduced HB thickness (F) in the Pgf  ^−/− HBs. Means with 95% confidence intervals are shown with P < 0.05, P < 0.01, and P < 0.001 represented by *, **, and *** respectively.

Figure 5

Whole mount vascular staining of circle of Willis (cW) at embryonic day (E) 14.5 and post-natal day (P) 7. Whole mount immunofluorescence with isolectin B4 (IB4; white) provided visualization of the anterior cerebral arteries (ACAs: white arrows), olfactory arteries (black arrows) and the anterior communicating artery (AComA; white arrowhead) in B6 and Pgf  ^−/− brains at E14.5 (A) and P7 (not shown). In many B6 brains, an extra vessel along the ACA was present (asterisk) while in some Pgf  ^−/− brains, the AComA was absent (black arrowhead). Significant changes in the Pgf  ^−/− cW included a narrower diameter of one ACA (B) but not both ACAs, resulting in a decreased ratio of the ACA diameters (C). The number of vessels present in the anterior cW of Pgf  ^−/− mice was fewer than in B6 mice. The mean number of AComAs in the Pgf  ^−/− cW at E14.5 was fewer than in B6 (D). Similarly, at E14.5, the mean number of collateral vessels in the Pgf  ^−/−cW was reduced (E). Means with 95% confidence intervals are shown with P < 0.05, P < 0.01, and P < 0.001 represented by *, **, and *** respectively.

Figure 6

Ink perfusion and imaging of the adult circle of Willis. The circle of Willis was imaged in adult male and female B6 and Pgf  ^−/− mice (A). The average number of anterior communicating arteries (AComAs) (arrowhead) was decreased in female Pgf  ^−/− mice (B). There was no significant difference in the number of anterior collateral vessels on the anterior cerebral arteries (arrow) in either male or female Pgf  ^−/− mice (C). Means with 95% confidence intervals are shown with P < 0.05 represented by *.

Figure 7

Infarct size and blood flow after left common carotid artery (LCCA) occlusion. Staining revealed no apparent infarct in B6 brain slices (A) but infarcted tissue (pale area encircled) in 2 female Pgf  ^−/− mice (B). The percentage of cerebral blood flow remaining after LCCA was significantly decreased in the Pgf  ^−/− mice (C). * represents P < 0.05.