CR6-interacting factor 1 deficiency impairs the activity of vascular endothelial nitric oxide synthase by inhibiting biosynthesis of tetrahydrobiopterin

Abstract

Background: CR6 interacting factor 1 (CRIF1) deficiency has been reported to impair vascular function by reduced activity of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production in endothelial cells. Tetrahydrobiopterin (BH4) is a key regulator of eNOS in cardiovascular diseases. Objective: The purpose of this study is to investigate whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling. Methods: Both CRIF1-deficient human umbilical vein endothelial cells and lung endothelial cells from CRIF1 knockout mice were assayed for BH4-associated proteins and enzymes. Results: Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with N-acetyl-L-cysteine treatment replenished BH4 levels by elevating levels of GCH1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. Conclusion: This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.