Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor

Abstract

Aberrant blood vessel growth in the retina that underlies the pathology of proliferative diabetic retinopathy and retinopathy of prematurity is the result of the ischemia-driven disruption of the normally antiangiogenic environment of the retina. In this study, we show that a potent inhibitor of angiogenesis found naturally in the normal eye, pigment epithelium-derived growth factor (PEDF), inhibits such aberrant blood vessel growth in a murine model of ischemia-induced retinopathy. Inhibition was proportional to dose and systemic delivery of recombinant protein at daily doses as low as 2.2 mg/kg could prevent aberrant endothelial cells from crossing the inner limiting membrane. PEDF appeared to inhibit angiogenesis by causing apoptosis of activated endothelial cells, because it induced apoptosis in cultured endothelial cells and an 8-fold increase in apoptotic endothelial cells could be detected in situ when the ischemic retinas of PEDF-treated animals were compared with vehicle-treated controls. The ability of low doses of PEDF to curtail aberrant growth of ocular endothelial cells without overt harm to retinal morphology suggests that this natural protein may be beneficial in the treatment of a variety of retinal vasculopathies.

Figure 1

In situ angiogenesis and apoptosis of endothelial cells in PEDF-treated retinas. (A) Examples shown at low (Left) and high (Right) power of retinas of mice exposed to hyperoxia and subsequently treated with vehicle (Aa–Ad), with 11.2 μg/day PEDF (Ae and Af) or with 22.4 μg/day PEDF (Ag and Ah). (B) Sections of retinas from mice treated with vehicle (Ba) or with 11.2 μg/day PEDF (Bb) were stained with PECAM-1 to detect endothelial cells (Ba1 and Bb1) and with TUNEL to detect apoptotic cells (Ba2 and Bb2), and the images merged (Ba3 and Bb3). L indicates the position of the lens. Solid arrows are reference points within each retina. The open arrowhead in panel Ba1–Ba3 points to a vascular tuft of predominantly TUNEL-negative endothelial cells. Note that the retinal pigment epithelial layer autofluoresces.

Figure 2

Effect of PEDF treatment on hyperoxia-induced retinal neovascularization. Mice exposed to hyperoxia to induce retinopathy (experiment 1, circles; experiment 2, triangles) and control mice maintained in ambient room air (squares) were treated systemically for 5 days with PEDF, and the endothelial cells at or beyond the internal limiting membrane (ILM) counted. (Error bars, SEM.)

Figure 3

PEDF induction of endothelial cell apoptosis in vitro. Increasing concentrations of PEDF were added to human microvascular endothelial cells and the proportion of apoptotic cells scored 18 h later. (Error bars, SEM.)