Nonischemic cerebral venous hypertension promotes a pro-angiogenic stage through HIF-1 downstream genes and leukocyte-derived MMP-9

Abstract

Cerebral venous hypertension (VH) and angiogenesis are implicated in the pathogenesis of brain arteriovenous malformation and dural arteriovenous fistulae. We studied the association of VH and angiogenesis using a mouse brain VH model. Sixty mice underwent external jugular vein and common carotid artery (CCA) anastomosis (VH model), CCA ligation, or sham dissection (n=20). Hypoxia-inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) and stromal-cell-derived factor-1alpha (SDF-1alpha) expression, and matrix metalloproteinase (MMP) activity were analyzed. We found VH animals had higher (P<0.05) sagittal sinus pressure (8+/-1 mm Hg) than control groups (1+/-1 mm Hg). Surface cerebral blood flow and mean arterial pressure did not change. Hypoxia-inducible factor-1alpha, VEGF, and SDF-1alpha expression increased (P<0.05). Neutrophils and MMP-9 activity increased 10-fold 1 day after surgery, gradually decreased afterward, and returned to baseline 2 weeks after surgery. Macrophages began to increase 3 days after surgery (P<0.05), which coincided with the changes in SDF-1alpha expression. Capillary density in the parasagittal cortex increased 17% compared with the controls. Our findings suggest that mild nonischemic VH results in a pro-angiogenic stage in the brain by upregulating HIF-1 and its downstream targets, VEGF and SDF-1alpha, increasing leukocyte infiltration and MMP-9 activity.

Figure 1. SSP pressure increased significantly in the VH brain

(A) Circulation of normal mouse brain. (B) Circulation of VH brain shows that CCA-EJV fistula (AVF) in the neck arterialized and pressurized the venous system. Arrows show direction of blood flow. (C) Photograph shows CCA-EJV anastomosis. SCM: Sternocleinomastoid muscle. Bar=1mm. (D) SSP. Creation of CCA-EJV fistula resulted in immediate and sustained increase of SSP. VH mice had constantly higher SSP pressure (8±1 mmHg) compared to CCA-ligation (1±1 mmHg, p<0.05) and sham groups (1±1 mmHg, p<0.05). (E) and (F) show that no significant changes for CBF and MBP were found pre- and post-operation in each group.

Figure 2. Increased HIF-1, VEGF and SDF-1 expression in the VH brain

(A) Photomicrographs show that HIF-1α, VEGF or SDF-1 expression was detected in the VH brain, but not in control brains (mice with CCA ligation). The expression of HIF-1α-, VEGF- or SDF-1-positive staining was localized to the endothelial cells (lectin staining, green) in vessel walls. The nuclei were counterstained with DAPI (blue). Scale Bar =20 μm. (B) Western blot analysis shows that VEGF expression increased in the VH brain. Image at left shows a representative Western blot. Minimal expression of VEGF was observed in the control group (CCA-ligation 1 week). Bar graph at right shows that VEGF expression increased 3 days and peaked 1 week after surgery (*p<0.05, compared with the CCA-ligation group). (C) Bar graph shows the results of ELISA analysis. The expression of SDF-1α began to increase at day 3, peaked at 1 week, and decreased 2 weeks post-operation. (*p<0.05, compared with the sham and CCA-ligation group)

Figure 3. Increased leukocytes in the VH brain

(A) Photomicroimage of MPO-stained sections of the brains of VH (b and d) and control (a and c) groups. One day after surgery, the MPO positive signals (brown color) increased significantly in VH brains (d), which were mainly located in the parasagittal cortex (CTX). Enlarged pictures from the selected areas (d) show that neutrophils were mainly located in the vascular wall. The nuclei were counterstained with hematoxylin. Neutrophils were absent in the brain tissue from the control groups (a and c). * in c indicates a similar-sized vessel in d. Scale bars for a and b are 1000 μm; for c and d, 50 μm. (B) Bar graph shows neutrophil counting. (*p<0.05, compared with the control group). (C) Increased expression of MMP-9 in the VH brain illustrated by gelatin zymogram gel image. S: MMP standards. Low level of MMP-9 activity was detected in the brains of control groups (Sham and CCA ligation) at day 1 after surgery. MMP-9 activity greatly increased in the VH brains day 1 after the surgery. (D) Bar graph shows the time course of MMP-9 expression. MMP-9 activity was highest at day 1 post operation, and then returned to the baseline 2 weeks after surgery. The time course of MMP-9 expression coincided with the infiltration of neutrophils. MMP-2 was expressed equally in all the groups at all time points. The control sample is from CCA-ligation group. (*p<0.05, compared with the control groups). (E) Photomicroimage shows macrophage infiltration in the parasagittal cortex of the VH brain 3 days after surgery. CD68 positive cells stained in brown color. There were few macrophages in CCA-ligation brains (a), whereas many macrophages were detected either in the vessel wall or in the parenchyma of VH brains (b). The nuclei were counterstained with hematoxylin. Scale bar = 40 μm. (F) Double fluorescence staining of CD68 (red) and Lectin (green) show that macrophage was localized in the vessel wall or in parenchyma. The nuclei were counterstained with DAPI. (G) Bar graph shows quantification of macrophages at different time points. Macrophages began to increase at day 3, and gradually increased and plateaued 1 week following surgery. The time course of macrophage infiltration coincided with increased SDF-1 expression. (*p<0.05, compared with the sham or ligation group)