Targeting neovascular pericytes in neurofibromatosis type 1

Abstract

Apart from tumor-driven neovascularization, a less-appreciated consequence of neurofibromatosis type 1 (NF1) is the hyperproliferation of vascular mural cells (pericytes). This study aims at establishing a role for pericytes in NF1, and determining whether interference with the function of a key pericyte component (NG2 proteoglycan) inhibits NF1 tumor neovascularization. Neovascularization in NF1 was studied in Nf+/+(control), Nf1+/-, and Nf1-/-embryos at E-10, ischemia-induced retinal angiogenesis model in 24 eyes of Nf1+/-, Nf1+/+mice, and in malignant peripheral nerve sheath tumors (MPNSTs) derived from NF1 patients (ST88-14, NMS-2PC) orthotopically grown in nude mice (Crl: nu/nu). The anti-angiogenic effect of intracorneal polymer pellets containing anti-NG2 neutralizing antibody was quantified in the nude-mouse corneal angiogenesis model in which angiogenesis was induced by xenografting NMS-2PC tumor into the corneal stroma of 22 eyes. By using confocal microscopy, immunohistochemistry, and BrdU proliferation assay, the pericyte/endothelium ratios and proliferation rates were measured. Activated pericytes were present at the leading tip of the angiogenic sprouts. Pericytes showed continuous investment of endothelium in both NMS-2PC and ST88-14 MPNST tumor xenografts. Mean corneal angiogenesis induced by NMS-2PC tumor grafts in NG2-antibody treated eyes was 1.491 and 3.186 mm2 in isotype-matched non-immunoglobulin treated eyes (control) (P=0.0002). A total of 193.8 vascular nuclei (a measure of ischemia-induced retinal angiogenesis) was present in angiogenic retinal tufts in Nf1+/- mice compared to 89.23 in Nf1+/+ mice (control) (P<0.0001). Mean pericyte/endothelium investment ratios were 1.015, 1.380, and 2.084 in control, Nf1+/-, and Nf1-/-embryos, respectively. Pericytes were 23% (control), 49% (Nf1+/-), and 69% (Nf1-/-) BrdU-positive. Endothelial cells from the same embryos were 29% (control), 47% (Nf1+/-), and 62% (Nf1-/-) BrdU-positive. Angiogenesis is accelerated in NF1 due to hyperproliferation of pericytes and endothelial cells. Mitotically activated NG2-positive pericytes, and endothelial cells may serve as potential therapeutic targets in NF1.