303A cell-Autonomous role for endothelial GTP cyclohydrolase 1 and tetrahydrobiopterin in blood pressure regulation.
Chuaiphichai S., Mcneill E., Douglas G., Crabtree M., Bendall J., Hale A., Alp NJ., Channon K.
Nitric oxide (NO), produced by endothelial NO synthase (NOS) is a key regulator of vascular tone and blood pressure. Tetrahydrobiopterin (BH4) is an essential cofactor for endothelial NOS. When BH4 levels become limiting, eNOS produces superoxide anion rather than NO and in turn further reduces NO bioavailability, resulting in endothelial dysfunction. Endothelial cell BH4 has been hypothesized to be critical in maintaining vascular function, but to date in vivo models of BH4 deficiency have achieved only partial global depletion of BH4 synthesis. We have investigated the specific role of endothelial cell BH4 using a novel line of conditional knockout mice. Mice homozygous for a floxed GCH1 (GCH1fl/fl) allele (encoding for GTPCH protein an essential enzyme in BH4 biosynthesis) were crossed with Tie2cre transgenic mice to produce GCH1fl/flTie2cre line, where the GCH1 gene is knocked out specific in endothelial cells. We have shown that the GCH1 gene is completely excised in isolated endothelial cells, with an accompanying lack of BH4 production in aortas from GCH1fl/flTie2cre mice. To determine the effects of endothelial cell BH4 deficiency on NO producing activity, we measured NO bioactivity by three complementary methods, each indicating deficient, or absent endothelial NO bioactivity in GCH1fl/flTie2cre mice compared with wild-type littermates. Superoxide production assessed by dihydroethidium HPLC was significantly 4-fold greater in primary endothelial cells from GCH1fl/flTie2cre mice compared with wild-type controls, which inhibited by L-NAME. Vasomotor studies demonstrated that GCH1fl/flTie2cre aortas and mesenteric arteries had significantly enhanced vasoconstriction to phenylephrine and impaired endothelium-dependent vasodilatations to acetylcholine and SLIGRL. The NOS-derived vasodilator in GCH1fl/flTie2cre aortas was identified as H2O2 using the scavenger PEG-catalase, which inhibited vasodilatation only in GCH1fl/flTie2cre aortas. Ex vivo supplementation of aortic rings with the BH4 analogue sepiapterin abolished NOS-derived H2O2 production and restored normal endothelial function in GCH1fl/flTie2cre aortas. GCH1fl/flTie2cre mice had higher systemic blood pressure than wild-type littermates (96.4±0.8 mmHg vs 103.4±1.3 mmHg; P<0.01), which was normalised by L-NAME. Taken together, these studies reveal an endothelial cell-autonomous requirement for GCH1 and BH4 in regulation of vascular tone and blood pressure, and identify endothelial cell BH4 as a pivotal regulator of NO vs. H2O2 as alternative eNOS-derived endothelial derived relaxing factors.