ARVO Presentation_Project Poster

1. Neovascular Tufts are Perfused Vascular Structures Formed
In Part by Macrophages and Microglia
Mauricio Rosenfeld, 1
Faith H. Barnett, 1
Edith Aguilar, 1
Martin Friedlander 1, 2
1
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA.
2
The Lowy Medical Research Institute, La Jolla, CA.
Contact and Funding
Email: mrosen@scripps.edu
National Eye Institute to MF (EY11254 ) and the Lowy Medical Research Institute.
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• Cells expressing the macrophage marker CD11B are structural components of
neovascular tufts in the OIR model.
• Tufts perfused by systemically injected dye demonstrate a functional connection
to the superficial vascular plexus in the neonatal mouse retina after OIR.
• Mac/Mg are structurally involved in the formation of perfused, multinucleated
tufts in both tumors and pathological angiogenesis in the eye.
Conclusions
Results
Lectin
R. Dextran
CD11b
Hoechst
Reference
1. Yoshiaki Kubota, Keiyo Takubo, Takatsune Shimizu, Hiroaki Ohno, Kazuo Kishi, Masabumi Shibuya, Hideyuki
Saya, and Toshio Suda. M-CSF inhibition targets pathological angigensis and
lymphangiogenesis. J. Exp. Med. 2009; 205(5): 1089-1102.
2. Smith LE, Wesolowski E, McLellan A, Kostyk SK, D'Amato R, Sullivan R, D'Amore PA. Oxygen-induced
retinopathy in the mouse. Invest Ophthalmol Vis Sci. 1994; 35(1): 101-11.
LSM Image
3D
IMARIS
After hi O2 induced central vascular obliteration, large and perfused multinucleated retinal
neovascular tufts are detectable in areas of the retina where the superficial vascular plexus
remains present.
This OIR P17 retinal Tuft is a perfused vascular structure. Rhodamine Lectin was administered by
intracardiac injection and retinal whole mount was not stained with antibody. The tuft’s body projects
towards the vitreous while connected to the superficial vascular plexus by way of a stalk-like structure.
Macrophages/Microglia cells (CD11b+
) are part of the structural composition of a retinal neovascular
tuft. These images represent a region of an OIR-P17 retinal superficial vascular plexus (lectin+
) as it
undergoes neovascularization.
HistologyBackground
During murine postnatal development, macrophages participate in
retinal vascular remodeling and pathological neovascularization in
oxygen–induced retinopathy 1
.
Recently, we have found that macrophages form a perfused
vascular mimicry (VM) network under hypoxic conditions in both
the subcutaneous matrigel angiogenesis model and in a mouse
melanoma tumor model. In the subcutaneous tumor model,
macrophages form perfused tuft-like structures (tumor rosettes)
within the tumor associated angiogenic vasculature.
Methods
For these studies we utilized the mouse OIR model described by
Smith et al 2
. Briefly, C57BL6/J mice were exposed to 75% O2 from
P7-P12, after which mice were returned to normal oxygen
conditions. This causes extensive central vaso-obliteration in the
retina followed by neovascularization, which is associated with
the formation of vascular tufts that extend toward the vitreous
from the superficial vascular plexus.
P17 mice received a 50ul intracardiac injection of a 50mg/ml Rhodamine Dextran
(155KD) solution in sterile saline. Dye was allowed to circulate for 4-5 minutes, mice
were euthanized, eyes enucleated and fixed. Retinal whole mounts were either left
untouched or stained with Lectin and a rat anti-mouse CD11B antibody. Confocal
microscopy was used to visualized neovascular tufts and Z-stack images were
gathered using a 63X oil immersion lens. 3D renderings of the original LSM images
were prepared using IMARIS software.
Hoechst CD11b CD31 Rhodamine Dextran
LSM Image
A B
Lectin CD11b Dextran Merge
LSM Image
3D
IMARIS
Figure A. Confocal image of neovascular tufts associated with the superficial vascular
plexus of a lectin stained OIR P17 retina whole mount.
Figure B. 3D rendition of a confocal retinal Z-stack depicting a small and perfused
multinucleated neovascular tuft in an OIR P17 retina. This particular tuft is positive
for both lectin and Cd11b.
A subcutaneous tumor bearing mouse was intravenously injected with low
molecular weight rhodamine dextran (3KD). Tumor tissue was harvested, fixed,
stained and imaged. CD11B+
Tumor associated macrophages form a perfused
VM network containing multinucleated neovascular rosettes that are therefore
connected to the traditional endothelial vasculature.
Purpose
Macrophages/microglia (Mac/Mg) are known to be closely
associated with retinal neovascular tufts during oxygen induced
retinopathy (OIR). To determine if the Mac/Mg were simply
juxtaposed to or structurally involved in these neovascular
structures, we studied the role of Mac/Mg in retinal neovascular
regions in a murine model of OIR.
lectin CD11b Merge
3D IMARIS
rosettes rosettes
*
*
*
*
*
*
*
*
*
*
stalk stalk
* *
**
stalk stalk
Primary Vascular Plexus
Original LSM
Primary Vascular Plexus Primary Vascular Plexus
Body of Tuft
Stalk
Body of Tuft
Stalk
IMARIS 3D IMARIS 3DVitreous VitreousVitreous
lectin CD11b Merge
Mergelectin
NV Tuft
NV Tuft
NV Tuft NV Tuft NV Tuft