Contribution of bone marrow-derived cells associated with brain angiogenesis is primarily through leukocytes and macrophages

Abstract

Objective: We investigated the role of bone marrow-derived cells (BMDCs) in an angiogenic focus, induced by VEGF stimulation.

Methods and results: BM from GFP donor mice was isolated and transplanted into lethally irradiated recipients. Four weeks after transplantation, groups of mice received adeno-associated viral vector (AAV)-VEGF or AAV-lacZ gene (control) injection and were euthanized at 1 to 24 weeks. BMDCs were characterized by double-labeled immunostaining. The function of BMDCs was further examined through matrix metalloproteinase (MMP)-2 and -9 activity. We found that capillary density increased after 2 weeks, peaked at 4 weeks (P<0.01), and sustained up to 24 weeks after gene transfer. GFP-positive BMDCs infiltration in the angiogenic focus began at 1 week, peaked at 2 weeks, and decreased thereafter. The GFP-positive BMDCs were colocalized with CD45 (94%), CD68 (71%), 5% Vimentin (5%), CD31/von Willebrand factor (vWF) (1%), and alpha-smooth muscle actin (alpha -SMA, 0.5%). Infiltrated BMDCs expressed MMP-9. MMP-9 KO mice confirmed the dependence of the angiogenic response on MMP-9 availability.

Conclusions: Nearly all BMDCs in the angiogenic focus showed expression for leukocytes/macrophages, indicating that BMDCs minimally incorporated into the neovasculature. Colocalization of MMPs with GFP suggests that BMDCs play a critical role in VEGF-induced angiogenic response through up-regulation of MMPs.

Figure 1

A. Lectin staining capillaries (red), and infiltrating BMDCs (green) in AAVlacZ and AAVVEGF groups after 1 to 24 weeks. Bar=100μm. B. The quantitative capillary density paralleling 1A. N=6/group, mean±SD, *=p<0.05, AAVVEGF vs. AAVlacZ group. C. Quantitative BMDCs at 0 to 24 weeks following AAVVEGF injection.

Figure 2

A. CD45 (upper) and CD68 (bottom) immunostaining in AAVVEGF-transduced brain. Arrows indicate CD45/GFP or CD68/GFP positive cells. Bar=50μm. B. GFP cells (green) and different cell markers (red). Yellow shows merged signals. Bar=25μm. C. Semi-quantitation of the percentage of BMDCs expressing different cell markers in AAVVEGF-transduced mice.

Figure 3

A. Diagram shows BMDCs (green) associated with ICAM-1 (red) expression in the ipsilateral side of VEGF-transduced brain. B. High magnification images (b, c, d) from boxed region in (a) indicate that BMDCs (b) associated with ICAM-1 (c), as shown in d. Bar=50μm.

Figure 4

Photomicrographs show MMP-9 (upper) and MMP-12 (bottom) immunostaining in the AAVVEGF-transduced brain. BMDCs (green) and MMPs (red) are well merged (arrows), suggesting that BMDCs express MMP-9 and MMP-12. Bar=25μm.

Figure 5

A. Lectin-stained capillaries in WT and MMP-9^-/- mice at 2 and 4 weeks after gene transfer. Bar=100μm. B. Quantitative capillary density parallels 5A. C. MMP-9 and MMP-2 activity (zymogram gel) in WT and MMP-9^-/- mice. D. Quantitative MMP-9 activity parallels 5C. N=6/group, mean±SD. *=p<0.05, AAVVEGF-transduced vs. AAVlacZ-transduced group (B, D).