Hypoxic oligodendrocyte precursor cell-derived VEGFA is associated with blood-brain barrier impairment

Abstract

Cerebral small vessel disease is characterised by decreased cerebral blood flow and blood-brain barrier impairments which play a key role in the development of white matter lesions. We hypothesised that cerebral hypoperfusion causes local hypoxia, affecting oligodendrocyte precursor cell-endothelial cell signalling leading to blood-brain barrier dysfunction as an early mechanism for the development of white matter lesions. Bilateral carotid artery stenosis was used as a mouse model for cerebral hypoperfusion. Pimonidazole, a hypoxic cell marker, was injected prior to humane sacrifice at day 7. Myelin content, vascular density, blood-brain barrier leakages, and hypoxic cell density were quantified. Primary mouse oligodendrocyte precursor cells were exposed to hypoxia and RNA sequencing was performed. Vegfa gene expression and protein secretion was examined in an oligodendrocyte precursor cell line exposed to hypoxia. Additionally, human blood plasma VEGFA levels were measured and correlated to blood-brain barrier permeability in normal-appearing white matter and white matter lesions of cerebral small vessel disease patients and controls. Cerebral blood flow was reduced in the stenosis mice, with an increase in hypoxic cell number and blood-brain barrier leakages in the cortical areas but no changes in myelin content or vascular density. Vegfa upregulation was identified in hypoxic oligodendrocyte precursor cells, which was mediated via Hif1α and Epas1. In humans, VEGFA plasma levels were increased in patients versus controls. VEGFA plasma levels were associated with increased blood-brain barrier permeability in normal appearing white matter of patients. Cerebral hypoperfusion mediates hypoxia induced VEGFA expression in oligodendrocyte precursor cells through Hif1α/Epas1 signalling. VEGFA could in turn increase BBB permeability. In humans, increased VEGFA plasma levels in cerebral small vessel disease patients were associated with increased blood-brain barrier permeability in the normal appearing white matter. Our results support a role of VEGFA expression in cerebral hypoperfusion as seen in cerebral small vessel disease.

Keywords: Angiogenesis; BBB; Glial biology; OPC; Vascular dementia; cSVD.

Fig. 1

Bilateral carotid artery stenosis led to a persistent decrease in cerebral blood flow. (A) LSCI images showing blood flow in superficial blood vessels captured at baseline, d0, and d7. Blood flow measurements in ROI1 (blue) contained all sizes superficial blood vessels, while ROI2 (red) contained the 3^rd order branch from superior sagittal sinus to avoid larger superficial vessels. Visualization of blood flow signal ranged from high flow (red) to low flow (blue). (B) Changes in CBF were quantified and compared to baseline measurements at d0 and d7 (ROI2). Scale bar, arbitrary value, 150 (low blood flow), 800 (high blood flow). Mean ± SEM; ns = not significant; #p < 0.05, ##p < 0.01 vs baseline measurements; **p < 0.01, vs Sham; unpaired student t-test

Fig. 2

BCAS led to an increase in hypoxic OPC in deep cortical regions after 7 days. (A) Immunolabeling for MBP and MBP clone SMI94 in the corpus callosum. Scale bar, 50 µm. (B) Quantification of MBP integrity, (C) MBP negative area, and (D) Myelin degradation, quantified by intensity signal and the number of hyperintense foci, respectively, was not statistically different between BCAS and Sham mice 7 post-operative days. (E) immunolabeling for Olig2, CC1, and Pimonidazole-Pacific blue in the deep cortical regions. Scale bar, 50 µm. (F) A significant increased hypoxic cell density was observed in the deep cortical regions of BCAS animals compared to Sham animals. (G) BCAS mice also showed an increased number of hypoxic OPC in these regions compared to Sham animals. Mean ± SEM; *p < 0.05, **p < 0.01; unpaired student t-test or Mann–Whitney U-test

Fig. 3

Hypoxia induced the expression of 417 DEGs. (A) Hypoxia (2% O₂) induced the expression of 417 differentially expressed genes (DEG) compared to normoxia (21% O₂) in OPC, of which 256 were upregulated and 171 downregulated. (B) Volcano plot showing the 417 DEG in hypoxic OPC with genes that were downregulated in red, and upregulated genes in green (C) DAVID enrichment analysis shows enrichment of pathways including HIF-1 signalling pathway, positive regulation of cell migration, and angiogenesis. Terms were considered significant with an EASE score < 0.05 and FDR < 0.05. (D) VEGFA was identified as a potential key mediator in these pathways and secreted protein concentration in the hypoxia exposed conditioned medium was increased compared to normoxia. Mean ± SEM; ****p < 0.0001, vs 21%; unpaired student t-test

Fig. 4

Hypoxia induced expression and secretion of VEGFA in Oli-neu cells. (A) The expression of Vegfa mRNA was constantly increased in hypoxic Oli-neu cells compared to normoxic cell expression from 12 to 48 h, with a peak increase after 24 h. (B) Hypoxic Oli-neu cells significantly secreted more VEGFA protein into conditioned media 24 h after exposure to hypoxia compared to normoxia. (C) Oli-neu cells exposed to hypoxia did not significantly increase mRNA expression of Hif1α after 24 h exposure, while Epas1 (aka Hif2α) was significantly increased compared to normoxic cell expression. (D) inhibition of Hif1α and/or Epas1 results in inhibition of hypoxia mediated increase in Vegfa expression. (E) Both Claudin-5 (Cldn5) and (F) Occludin (Ocln) mRNA expression was significantly decreased in brain EC treated with hypoxic OPC derived CM. Mean ± SEM; ns = not significant, **p < 0.01, ****p < 0.0001, vs 21%; unpaired student t-test or one-way ANOVA with Tukey’s multiple comparisons test

Fig. 5

BCAS led to an increase in BBB permeability after 7 days without changes in vascular density. (A) immunolabeling for lectin in the cortex, deep cortex, corpus callosum, and striatum. Scale bar, 50 µm. (B) Quantification of vessel density in BCAS compared to Sham. No statistical differences were found in vessel density when comparing BCAS to Sham in the respective areas. (C) Immunolabeling for blood vessel (Lectin), and mouse IgG for BBB leakages. Scale bar, 50 µm. (D) The number of leakages and (E) the total leakage size was significantly higher in BCAS compared to Sham. Mean ± SEM; **p < 0.01, vs Sham; unpaired student t-test

Fig. 6

Increased VEGFA plasma levels in cSVD patients. (A) VEGFA blood plasma levels in cSVD patients were higher compared to age and sex-matched controls. (B) There was no correlation between VEGFA plasma levels and WMH volume in patients or controls. A trend in the relation between VEGFA plasma levels and leakage rate in (C) WMH and (D) NAWM in patients, but not in controls, was observed (indicated by the solid for patients and dotted line for controls). Abbreviations: WMH = white matter hyperintensities; NAWM = normal appearing white matter; K_i = leakage rate. For quantification, mean ± SEM; *p < 0.05; Mann–Whitney U-test