We asked whether the hypoxia-regulated factor, insulin-like growth factor binding protein-3 (IGFBP3), could modulate stem cell factor receptor (c-kit ؉ ), stem cell antigen-1 (sca-1 ؉ ), hematopoietic stem cell (HSC), or CD34 ؉ endothelial precursor cell (EPC) function. Exposure of CD34 ؉ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygeninduced retinopathy (OIR) model. In separate studies, GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration, protection of the developing vasculature from hyperoxia-induced regression, and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion, IGFBP3 mediates EPC migration, differentiation, and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions. IGFBP3 ͉ angiogenesis ͉ retinopathy of prematurity V ascular damage associated with diabetic retinopathy and retinopathy of prematurity (ROP) results from tissue ischemia, and, subsequently, this ischemia leads to development of pathological neovascularization. Insulin-like growth factor 1 (IGF1) is required for normal retinal vascular development because vascular development is arrested in its absence despite the presence of VEGF (1). Development of ROP is associated with low levels of IGF1 (2) because the lack of IGF1 in the early neonatal period leads to the development of avascular retina, which results in ROP (3). However, unregulated IGF1 expression can lead to pathological neovascularization (4-13), and IGF1 receptor (IGF1R) antagonists are able to suppress retinal neovascularization in vivo by inhibiting VEGF signaling (1).The effects of IGF1 are mediated by IGF1R and modulated by complex interactions with IGF binding proteins (IGFBPs), which are also modulated at multiple levels. Six IGFBPs function as transporter proteins and storage pools for IGF1 in a tissue-and developmental stage-specific manner. Phosphorylation, proteolysis, polymerization (8), and cell or matrix association (9) regulates the functions of IGFBPs. Specific IGFBPs have been shown to either stimulate or inhibit IGF1 action (10).IGFBP3, the best studied and most abundant of these binding proteins, carries Ն75% of serum IGF1 and IGF2 in heterotrimeric complexes. Besides its endocrine effects, IGFBP3 has auto-and paracrine actions affecting cell mobility, adhesion, apoptosis, survival, and the cell cycle (14,15). Like the other IGFBPs, IGFBP3 has IGF1-in...

Rationale Insulin-like growth factor binding protein-3 (IGFBP-3) modulates vascular development by regulating endothelial progenitor cell (EPC) behavior, specifically stimulating EPC cell migration. This study was undertaken to investigate the mechanism of IGFBP-3 effects on EPC function and how IGFBP-3 mediates cytoprotection following vascular injury. Objective To examine the mechanism of IGFBP-3 mediated repair following vascular injury. Methods and Results We utilized two complementary vascular injury models: laser occlusion of retinal vessels in adult GFP chimeric mice and oxygen induced retinopathy (OIR) in mouse pups. Intravitreal injection of IGFBP-3 expressing plasmid into lasered GFP chimeric mice stimulated homing of endothelial precursor cells (EPCs), while reversing ischemia induced increases in macrophage infiltration. IGFBP-3 also reduced the retinal ceramide/sphingomyelin ratio that was increased following laser injury. In the OIR model, IGFBP-3 prevented cell death of resident vascular endothelial cells and EPCs, while simultaneously increasing astrocytic ensheathment of vessels. For EPCs to orchestrate repair, these cells must migrate into ischemic tissue. This migratory ability is mediated, in part, by endogenous nitric oxide (NO) generation. Thus, we asked whether IGFBP-3’s migratory effects were due to stimulation of NO generation. IGFBP-3 increased eNOS expression in human EPCs leading to NO generation. IGFBP-3 exposure also led to the redistribution of vasodilator stimulated phosphoprotein (VASP), a NO regulated protein critical for cell migration. IGFBP-3 mediated NO generation required HDL receptor activation and stimulation of PI3K/Akt pathway. Conclusion These studies support consideration of IGFBP-3 as a novel agent to restore the function of injured vasculature and restore NO generation.

This study was conducted to determine the perivascular cell responses to increased endothelial cell expression of insulin-like growth factor binding protein-3 (IGFBP-3) in mouse retina. The contribution of bone marrow cells in the IGFBP-3-mediated response was examined using green fluorescent protein-positive (GFP(+)) adult chimeric mice subjected to laser-induced retinal vessel occlusion injury. Intravitreal injection of an endothelial-specific IGFBP-3-expressing plasmid resulted in increased differentiation of GFP(+) hematopoietic stem cells (HSCs) into pericytes and astrocytes as determined by immunohistochemical analysis. Administration of IGFBP-3 plasmid to mouse pups that underwent the oxygen-induced retinopathy model resulted in increased pericyte ensheathment and reduced pericyte apoptosis in the developing retina. Increased IGFBP-3 expression reduced the number of activated microglial cells and decreased apoptosis of neuronal cells in the oxygen-induced retinopathy model. In summary, IGFBP-3 increased differentiation of GFP(+) HSCs into pericytes and astrocytes while increasing vascular ensheathment of pericytes and decreasing apoptosis of pericytes and retinal neurons. All of these cytoprotective effects exhibited by IGFBP-3 overexpression can result in a more stable retinal vascular bed. Thus, endothelial expression of IGFBP-3 may represent a physiologic response to injury and may represent a therapeutic strategy for the treatment of ischemic vascular eye diseases, such as diabetic retinopathy and retinopathy of prematurity.