BM-derived CX3CR1+ progenitors potentiate vascular repair in an ischemic retinopathy mouse model. Injection of myeloid progenitor cells may provide a potential clinical application for the treatment of retinal vascular disease. CX3CR1+/CD34+ BM cells migrated more to the retina compared to CX3CR1+/CD34- cells and decreased areas of vascular obliteration and neovascular tufts in an OIR mouse model. CX3CR1+/CD34+ cells also upregulated more proangiogenic genes than CX3CR1+/CD34- cells by qPCR analysis.

1. Bone
T H E
S C R I P P S
R E S E A R C H
I N S T I T U T E
Bone marrow derived CX3CR1+ progenitors facilitate vascular repair in a
murine model of ischemic retinopathy
Results
Acknowledgements
Conclusions
This work was supported by grants to M.F. from the National Eye Institute (RO1 EY011254)
and the Lowy Medical Research Institute.
Results (continued)
Edith Aguilar, Susumu Sakimoto, Salomé Murinello, Peter D. Westenskow, Yoshihiko Usui, Felicitas Bucher, Maki Kitano,
Daniel Feitelberg, Mauricio Rosenfeld, Martin Friedlander
Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, CA
References
1. Smith LE, et al . Oxygen –induced retinopathy in the mouse. Invest Ophthalmol. Vis. Sci. 1994;
35:101-111.
2. Ritter MR et al. Myeloid progenitors differentiate into microglia and promote vascular repair in a model
of Ischemic Retinopathy. J Clin Invest. 2006 116:3266-76.
3. Banin E, et al.T2-TrpRS Inhibits Preretinal Neovascularization and Enhances Physiological Vascular
Regrowth in OIR as Assessed by a New Method of Quantification Invest Ophthalmol Vis Sci. 2006
May;47(5):2125-34.
4. Marchetti V,et al. Differential macrophage polarization promotes tissue remodeling and repair in a
model of ischemic retinopathy. Sci Rep. 2011;1:76.
5. Ginhoux F, et al. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat
Rev Immunol. 2014;14:392-404.
6. Kumar AH, et al. Bone marrow derived CX3CR1 progenitors contribute to neointimal
smooth muscle cells via fractalkine CX3CR1 interaction. FASEB J. 2010; 24: 81-92.
BM-derived CX3CR1+ progenitors potentiate vascular repair in an ischemic retinopathy
mouse model. Injection of myeloid progenitor cells may provide a potential clinical
application for the treatment of retinal vascular disease.
Bone marrow (BM) derived cells have been reported to serve as
proangiogenic microglia or macrophage in various ischemic
conditions. We previously reported a subpopulation of myeloid
progenitor cells that differentiate into microglia. These cells
promoted vascular repair in oxygen-induced retinopathy (OIR). In
this study, we wanted to further characterize the differentiation
state of these myeloid progenitor cells.
Purpose
Figure 4 . Flow Cytometry analysis. 3.1 % of BM cells were CX3CR1+/CD34+ and
2.3 % were CX3CR1+/CD34-.
Microglia and/or macrophages play an important role in the
formation of the normal superficial and intermediate retinal
vascular plexuses by releasing pro-angiogenic factors. The
chemokine receptor CX3CR1 is associated with the
monocyte/macrophage/DC lineage, while CD34 is associated with
their progenitor cells. In this study, we show that intravitreal
injections of undifferentiated CX3CR1+/CD34+ BM cells migrate to
the retina and facilitate normalization of retinal vasculature in OIR.
Methods
Figure 3. C57Bl/6J mice were exposed to the oxygen-induced
retinopathy model (OIR). Animals were subjected to 75%
oxygen from P7 to P12, injected with fluorescently labeled
cells at P7, and subsequently analyzed at P17.
Intravitreal Injection:
Right Eye: CX3CR1+/CD34+ cells
Left Eye: CX3CR1+/CD34- cells
1.0x105 cells / eye
P7 P12 P17
Hyperoxia (75% O2) NormoxiaNormoxia
Introduction
Figure 5. Continued
HSC GMP MDP
Common Monocyte
Progenitor
Monocyte
↓
(Macrophage, DC)
CD34
CX3CR1
HSC: Hematopoietic Stem Cells
GMP: Granulocyte-macrophage progenitor
MDP: Macrophage and Dendritic Cell (DC) precursor
Control Lin- HSC Ritter et al. 2006
CX3CR1+ CD34+
2.99%
CX3CR1+ CD34-
6.23%
Bone Marrow
isolation
CX3CR1
CD34
CX3CR1+ CD34+CX3CR1+ CD34-
Isolectin-B4
Bone Marrow (BM) cells from 6 weeks old transgenic CX3CR1GFP/+ mice were
isolated by flow cytometry.
C57Bl/6J OIR mice were sacrificed at P17, evaluated by whole mount preparation,
and stained with Isolectin Griffonia Simplicifolia I-B4 Alexa Fluor 568 (Invitrogen).
We quantified the areas of pathological neovascularization and obliteration as
previously described (Banin et al 2006).
Also we performed RT-PCR with an array of 84 angiogenesis-related genes for
CX3CR1+CD34+ cells and CX3CR1+CD34-.
Figure 5 . (A) In the OIR model, injected CX3CR1+/CD34+ BM cells homed to the retinal
vessels more than CX3CR1+/34-; and (B and C) injection of CX3CR1+/CD34+ BM cells
decreased both the area of vascular obliteration and neovascular tufts compared to
CX3CR1+/CD34- BM cells injection (*p=0.019 and p=0.018, respectively). Bar 500um.
Methods (continued)
A
B
C
CX3CR1+
CD34+
CX3CR1+
CD34-
Figure 6 . Results of qPCR based array analysis showing angiogenic gene upregulated in
CX3CR1+/CD34+ BM cells compared to CX3CR1+/CD34-.
CX3CR1Isolectin-B4
CX3CR1+/CD34+ Injected OIR
* *
0
2
4
6
8
10
12
ANGPT1 ENG F3 MMP14 PF4 PLG SPHK1 TIE1 TYMP VEGFA
CX3CR1+CD34- CX3CR1+CD34+
CX3CR1+
CD34+
CX3CR1+
CD34-
CX3CR1+
CD34+
CX3CR1+
CD34-
Figure 2 .
Figure 1 .
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