Tetrahydrobiopterin (BH4) deficiency is associated with microglial activation and microvascular degeneration in the eye.

Abstract

Purpose: Tetrahydrobiopterin (BH4) is an essential cofactor in multiple metabolic processes and plays an essential role in maintaining the inflammatory and vascular homeostasis. In this study, we have investigated the deleterious effects of BH4 deficiency on retinal vasculature during development.

Methods: hph-1 mice (C57BL/6xCBA), which display 90% deficiency in guanine triphosphate cyclohydrolase (GTP), the rate-limiting enzyme in BH4 synthesis, were used to characterize the inflammatory effects of BH4 deficiency and the integrity of retinal vessels in the eye. BH4 levels were measured by HPLC in the retinas from hph-1 and wild type mice. The integrity of the inner and superficial retinal vasculature and number of microglia cells was evaluated at different ages in both groups of animals on retinal flat-mounts stained with lectin and Iba-1. Retinal expression of inflammatory factors such as NRLP-3, interleukin-1 beta (IL-1b), and interleukin-6 (IL-6), as well Norrin and its receptor Frizzled-4 (Fz4) strongly involved in the control of retinal vascular development, were analyzed by qPCR.

Results: Our findings demonstrated that BH4 levels in the eye were significantly decreased in hph-1 animals by ~50% compared to wild type mice. The eyes from hph-1 mice showed a decreased size at all the ages analyzed compared to WT animals. Interestingly, hph-1 mice at postnatal day 22 (P22), showed iris atrophy, persistent fetal vasculature, significant increase in the number of microglial cells (p<0.01), as well as a marked degeneration of the inner retinal microvasculature. Augmented expression of pro-inflammatory factors, NRLP-3 (3.6-fold), IL-1b (12-fold) and IL-6 (2.9-fold) and decreased expression of Norrin (0.2-fold) and its receptor FZ4 (0.7-fold) also were associated with a deficiency of BH4 in the retina of the hph-1 mice at P22.

Conclusions: Our data reveal that BH4 is an important cofactor involved in eye development and its deficiency results in a robust retinal inflammation associated with microvasculature dysfunction that impairs the normal function of the eye.