Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy

Abstract

Vision loss associated with ischemic diseases such as retinopathy of prematurity and diabetic retinopathy are often due to retinal neovascularization. While significant progress has been made in the development of compounds useful for the treatment of abnormal vascular permeability and proliferation, such therapies do not address the underlying hypoxia that stimulates the observed vascular growth. Using a model of oxygen-induced retinopathy, we demonstrate that a population of adult BM-derived myeloid progenitor cells migrated to avascular regions of the retina, differentiated into microglia, and facilitated normalization of the vasculature. Myeloid-specific hypoxia-inducible factor 1alpha (HIF-1alpha) expression was required for this function, and we also demonstrate that endogenous microglia participated in retinal vascularization. These findings suggest what we believe to be a novel therapeutic approach for the treatment of ischemic retinopathies that promotes vascular repair rather than destruction.

Figure 1. Retinal vascular development in normal and OIR mice.

The mouse is born with a largely avascular retina (A and B). During the first postnatal week, superficial retinal vessels grow radially from the optic nerve head (C). The deep retinal vasculature is established through branching of the superficial layer during the second week (D). A third intermediate plexus of vessels forms, and the mature retinal vasculature is established at around P30 (E and F). Exposure to hyperoxia causes central vaso-obliteration (G), and after returning to normoxia at P12, characteristic preretinal neovascular tufts form at the interface between the vascularized (peripheral) and avascular (central) retina (H). (I–N) Lin^–HSCs promote vascular repair in the OIR model. Lin^–HSC injected intravitreally prior to oxygen exposure dramatically accelerated revascularization compared with the vehicle-treated fellow eye at P17. While retinas treated with vehicle showed partial absence of the superficial vasculature (I) and complete absence of the deep retinal vasculature (K and M), the Lin^–HSC-treated eye showed relatively normal retinal vasculature (J, L, and N). (O) A dramatically higher proportion of eyes treated with Lin^–HSC were fully revascularized at P17 compared with control eyes. Vessels were visualized by cardiac perfusion of fluorescein-dextran in A–F, I, and J (B, D, and F are images taken from 3D renderings rotated 90 degrees), and by GS lectin in G, H, and K–N. Nuclei in K–N were labeled with DAPI (blue). RE, right eye; LE, left eye. Magnification, ×4 (A, C, E, G, and H), ×10 (I and J), ×60 (B, D, and F).

Figure 2. BM subpopulations accelerate retinal revascularization and reduce preretinal neovascular tuft formation in OIR.

(A–D) Computer-assisted image analysis was used to calculate the area of retinal vessel obliteration (yellow) and preretinal neovascular tuft formation (red) in whole mounts from OIR eyes at P17 (17). (E) Retinas treated with Lin^–HSC prior to hyperoxia showed an almost 6-fold reduction in obliterated area versus uninjected controls and an approximately 5-fold reduction compared with vehicle-treated controls. (F) Lin^–HSC treatment significantly reduced neovascular tufts compared with uninjected and vehicle-treated eyes. (G) Lin^–HSCs reduced obliteration when administered prior to hyperoxia and during hyperoxia (P9 or P11) or just after return to normoxia (P12). inj, injected. (H) Mouse BM contained CD44^hi and CD44^lo fractions and the Lin^–HSC population was enriched for CD44^hi cells (red). Insets: Light-scattering properties of the CD44^hi cells were typical of monocytes and granulocytes, while those of CD44^lo cells were typical of lymphocytes. (I and J) Representative P17 retinas from eyes treated with CD44^lo and CD44^hi BM cells at P7. (K and L) Areas of obliteration (yellow) and neovascularization (red) at P17. When treated at P7, vascular obliteration (M) and neovascularization (N) were reduced in eyes treated with CD44^hi cells with efficacy similar to eyes treated with Lin^–HSC cells. Obliteration was similar between eyes treated with Lin^–HSC and CD44^hi cells, and neovascularization did not differ significantly between these groups (P = 0.25). Values represent mean ± SEM. *P < 10^–5; **P ≤ 0.006. Magnification, ×4.

Figure 3. The CD44^hi subpopulation expresses markers and genes characteristic of myeloid cells.

Two-color flow cytometry was used to characterize CD44 populations in whole mouse BM. All cells were labeled with an antibody against CD44 and colabeled with the indicated antibodies. CD44^hi cells are distinguished from CD44^lo and CD44^– cells by the horizontal lines in each plot. The CD44^hi population showed strong labeling for CD11b and Ly6G/C. Fractions of CD44^hi cells (to the right of the vertical lines) were positive for F4/80, CD14, and CD115. These antigens are present on cells of the myeloid lineage (–42). Positive labeling for CD11a and cKit was also observed on CD44^hi cells. CD44^lo cells labeled strongly with Ter119 and CD45R/B220 which are markers for erythroid and lymphoid cells, respectively (43, 44).

Figure 4. HIF-1α expression is required for myeloid progenitors to mediate repair in the OIR model.

(A and B) Representative GS lectin–stained retina whole mounts from eyes treated with CD44^hi cells from myeloid-specific HIF-1α knockout mice (A) or wild-type mice (B). (C and D) Quantified areas of vascular obliteration (yellow) and neovascular tufts (red) in retinas from A and B. (E) Compiled data showing significant loss of repair activity in the eyes treated with CD44^hi cells from HIF-1α knockouts. ^†P ≤ 0.0003, ^‡P = 0.024. Values represent mean ± SEM (n = 15). Statistics were generated using paired eyes. Magnification, ×4.

Figure 5. Intravitreally injected CD44^hi cells take on characteristics of retinal microglia.

(A) Transplanted CD44^hi cells (green) injected at P7 localized to and survived within the avascular central retina at P12. Many cells labeled with GS lectin (red), which labels microglia in the retina, and thus appear yellow. (B) CD44^hi cells (green, arrowhead) often assumed a ramified morphology similar to endogenous microglia. Endogenous microglia (arrow) and retinal vessels were stained with GS lectin (red). (C–F) In addition to the intervascular localization shown in B, transplanted CD44^hi cells also assumed a perivascular localization and were positive for F4/80 (C) and CD11b (E), markers of macrophages/microglia. D and F are merges with GFP expression (green), GS lectin (blue), and F4/80 (red, D) or CD11b (red, F). (G) CD44^hi cells assumed both perivascular and intervascular localization (3D image). Note the ramified, microglia-like morphology of the GFP⁺ cells. (H and I) CD44^hi cells did not form any portion of the vessel lumen, as shown by 3D imaging. The image in H was rotated such that the vessel could be viewed in cross-section. (I) Numbered positions indicated in H show that the GFP⁺ cell (green) was found on the outside of the CD31-labeled (dotted) endothelial lumen. (J and K) A single transplanted CD44^hi cell (3D image) shown en face (J) and in profile (K) demonstrating the highly ramified morphology taken on by these cells after injection into the eye. Magnification, ×10 (A), ×60 (B–I), ×120 (J and K).

Figure 6. Microglia participate in retinal vascularization.

Comparing C57BL/6J and BALB/cByJ strains revealed a difference in the number of CD11b⁺ microglia during the ischemic phase of OIR. (A) In whole mounts, both strains showed similar vaso-obliteration in the central retina at P12. However, at P17, there were dramatic differences in the vasculature, with C57BL/6J showing abundant neovascular tufts and little revascularization of the central retina. (B) C57BL/6J retinas contained fewer CD11b⁺ microglia over the course of 48 hours of ischemia compared with BALB/cByJ. The optic nerve head is positioned at the lower right in all images. (C) Quantification of CD11b⁺ microglia over time shows that C57BL/6J retinas had fewer microglia at P12 (0 hours) and less than half the number of microglia present in the retinas of BALB/cByJ mice at P14 (48 hours of ischemia). P ≤ 0.02 for BALB/cByJ versus C57BL/6J at all time points (n = 8–11). (D) Microglia depletion in C57BL/6J induced dramatic loss of preexisting microvasculature during retinal development. Injection of clodronate liposomes at P5 resulted in significant loss of CD11b⁺ microglia and capillary dropout at P8. Images depict similar locations in the central retina, with the optic nerve head at lower right. (E) Injection of clodronate liposomes into C57BL/6J at P2 caused significant depletion of CD11b⁺ microglia and dramatic disruption of the vasculature at P6 compared with control PBS liposome–treated fellow eye. Inset: Labeled liposomes (red) were taken up specifically by CD11b⁺ microglia (green) and not by GS lectin–labeled vascular cells (blue), nor any other CD11b^– cells. Magnification, ×4 (A and E, top panels), ×10 (D and E, bottom panels), ×20 (B), ×60 (E, inset).