The ARF Tumor Suppressor Is Essential for Vitreous Maturation and Hyaloid Vascular Regression

Abstract

Abstract: : Purpose: During mammalian eye development, the hyaloid vascular system (HVS) regresses as the primary vitreous matures. Failed HVS regression during vitreous maturation is central to the human eye disease Persistent Hyperplastic Primary Vitreous (PHPV). We have discovered that mice lacking the tumor suppressor gene Arf develop a PHPV-like pathology. We have begun to explore mechanisms by which Arf regulates vitreous maturation and HVS regression. Methods: Histological studies of eyes taken from embryonic day 16.5 through postnatal day 28 wild type and Arf-/- mice were performed to define the pathogenesis of developmental abnormalities in the eyes of Arf-null mice. Cultured mouse pericyte-like cells were used to begin to elucidate cellular and molecular mechanisms for Arf-dependent effects. Results: We observed that neonatal Arf-null mice have microphthalmia; retrolental tissue containing elements of the HVS; posterior lens capsule destruction with lens degeneration and cataract formation; and retinal detachment and dysplasia. These abnormalities, closely mimicking clinical and pathological aspects of PHPV, were completely penetrant in Arf-/- mice (n = 32 eyes) and absent in Arf+/- (n = 11 eyes) and wild type littermates (n = 15 eyes). The principal defect in Arf-/- eyes, observed as early as embryonic day 16.5, was the excess accumulation of perivascular cells within the HVS. During embryonic and postnatal development, these cells were actively proliferating, as evidenced by the presence of mitotic figures and expression of Ki-67 proliferating cell nuclear antigen in Arf-/- mice. Arf mRNA was detectable in the vitreous until postnatal day 5 and appeared to be localized to pericyte-like cells within the HVS. Pericytes generate angiogenic factors, like vascular endothelial growth factor (VEGF), to maintain vascular integrity. To address whether Arf expression might arrest pericyte proliferation and repress VEGF, we used an in vitro model of cultured pericytes. Retrovirus-mediated Arf expression in these cells arrested proliferation in G₁ phase of the cell cycle and repressed the expression of VEGF. Conclusions: Arf expression in vitreous cells in the developing eye prevents excess perivascular cell proliferation and may limit the expression of VEGF in perivascular cells. This would result in a paucity of supportive perivascular cells and low expression of VEGF within the HVS as mechanisms to promote HVS regression during vitreous maturation. Our findings suggest that abnormalities in the human ARF gene or its regulators may contribute to PHPV pathogenesis.

Keywords: vitreous • transgenics/knock-outs • molecular biology