Stromal Derived Factor–1 Affects Multiple Steps in Stem Cell Recruitment to Areas of Ocular Neovascularization

Abstract

Abstract: : Purpose: Stromal derived factor–1 (SDF–1) plays a critical role in homing and recruitment of hematopoietic stem cells (HSC) and endothelial precursor cells (EPC) to areas of injury, where these cells participate in compensatory angiogenesis. Increased vitreal SDF–1 levels exist in patients with ocular NV. The SDF–1 receptor CXCR4 is expressed on proliferating endothelium of developing blood vessels. We sought to determine the mechanism by which SDF–1 may promote HSC/EPC incorporation into ocular NV. Methods: CD34⁺ circulating EPC were isolated from plasma of normal adults. Human retinal endothelial cells (HREC) were isolated and grown as previously described. VCAM–1 and occludin expression were measured by Western, VEGF by ELISA, chemotaxis by the Boyden chamber assay, and haplotaxis by wounding assay. Lethally irradiated adult C57BL/6J mice (n=12) were reconstituted with purified Lin^–Sca–1⁺ HSC from bone marrow of gfp^+/+ donor mice and retinal NV was induced as previously described. Anti–SDF–1 was injected intravitreally (n=6). Eyes were enucleated, and the neural retinas were flat–mounted and evaluated by confocal microscopy. Results: SDF–1 (100 nM) resulted in a 40% increase in VEGF, a 3–fold increase in VCAM–1, and a 3–fold decrease in occludin expression in HREC (p<0.05). SDF–1 induced a dose–dependent increase in EPC migration (up to 13–fold over control levels, with peaks at 0.1 nM and 100 nM [p< 0.001]). This effect on migration was blocked by anti–CXCR4 and by AMD 3100, a pharmacological inhibitor of CXCR4. SDF–1 (0.1 and 100 nM) increased HREC haplotaxis at 94 hrs (p<0.05). Intravitreal administration of anti–SDF–1 dramatically reduced retinal NV in the reconstituted mice. Conclusions: Our studies support that local SDF–1 expression may increase NV by increasing VEGF expression. SDF–1 appears to increase the interaction between EPC and resident endothelial cells by increasing endothelial cell VCAM–1 expression. This effect allows recruited EPC to attach to resident endothelium and participate in NV. Our studies support that SDF–1 may facilitate migration of EPC between endothelial cells of the established vessel by disrupting tight junction occludins. Our studies also support that SDF–1 may contribute to later steps of NV by facilitating REC migration. Blocking SDF–1 prevents retinal NV and may represent a new therapeutic target for proliferative retinopathies.

Keywords: neovascularization • cytokines/chemokines • vascular cells