Role of endothelial and macrophage tetrahydrobiopterin in development and progression of atherosclerosis: BH4 puzzle solved?

According to the Global Burden of Disease study, cardiovascular risk factors and diseases such as hypertension, ischaemic heart disease, smoking, and cerebrovascular disease are the leading triggers of global mortality and life years spent with severe disability. More recently, epidemiological evidence revealed that these traditional cardiovascular risk factors are further boosted by environmental risk factors including noise and air pollution that are associated with an increased incidence of cardiovascular disease and mortality. All of these traditional and novel cardiovascular risk factors will lead to endothelial dysfunction, a form of subclinical atherosclerosis (for review, see ref.), and, at the end, to full-blown atherosclerosis and subsequently acute coronary syndromes and heart failure. Oxidative stress is a hallmark or cardiovascular disease characterized by and/or secondary to an activation of the vascular, but also phagocytic NADPH oxidase and by an uncoupling of the endothelial nitric oxide synthase (eNOS). The eNOS uncoupling process includes a functional depletion of the eNOS cofactor tetrahydrobiopterin (BH4) via an oxidation of BH4 to the BH3 radical, 7 adverse phosphorylation at Thr495 and Tyr657, 8,9 S-glutathionylation of cysteines in the reductase domain, oxidative disruption of the zinc–sulfur dimer binding site, and redox regulation of asymmetric dimethylarginine formation and breakdown. eNOS uncoupling means a switch from an anti-atherosclerotic NO-producing enzyme to a superoxide-producing pro-atherosclerotic enzyme. This is why eNOS is also called a Janus-faced enzyme (see Figure 1 for summary). The essential eNOS cofactor BH4 is mainly synthesized by GTP-cyclohydrolase-1 (GTPCH), which is degraded upon oxidative activation of the 26S proteasome, leaving the ‘salvage pathway’ for recycling of dihydrobiopterin (BH2) to BH4 by dihydrofolate reductase (DHFR). 16 With their actual work, Douglas et al. demonstrate the important role of endothelial and myeloid (e.g. macrophage) BH4 for development and progression of endothelial dysfunction and atherosclerosis by using high-fat diet feeding of control with normal GTPCH expression but global ApoE knockout (Gch1ApoE) and endothelial/myeloid-specific GTPCH and ApoE global knockout (Gch1Tie2CreApoE) mice. Specific deletion of GTPCH in endothelial and myeloid cells had no significant effect on body weight, blood lipids, circulating white blood cell count, or heart rate in both mouse strains with either normal or high-fat diet. Tie2specific deletion of GTPCH almost completely abolished GTPCH gene and protein expression in aorta, isolated endothelial cells or bone marrow-derived macrophages, which was associated with significantly lower BH4 levels, and increased superoxide formation rates in these cells or aorta. Of note, aortic BH4 levels were increased in high-fat diet-fed atherosclerotic control mice when compared with the normal diet group, whereas BH4 levels showed a decrease in all GTPCH-deficient groups regardless of the feeding protocol. The latter observation was less clear for circulating BH4 levels in plasma, as explained by the presence of significant GTPCH levels in liver tissue and generation of hepatic BH4 in both mouse strains. Systolic blood pressure was moderately increased by GTPCH deficiency, regardless of the feeding protocol (except in female high-fat dietfed mice), in line with a significant degree of endothelial dysfunction in Gch1Tie2CreApoE mice that became more pronounced upon highfat diet feeding and was rescued by ex vivo administration of sepiapterin. GTPCH deficiency also resulted in larger plaque areas, increased macrophage content in these plaques, and higher expression levels of Vcam-1, TNFa, and eNOS (Ccl2 by trend) in the high-fat diet groups, all of which was mirrored by suppressed inducible nitric oxide synthase (iNOS) activity in isolated bone marrow-derived macrophages upon chemotactic stimuli despite unaltered iNOS expression levels. GTPCH is constitutively expressed in macrophages but substantially induced by proinflammatory cytokines in accordance with the concept that the several 100-fold upregulation of iNOS upon these stimuli requires much higher concentrations of BH4 for its proper function, as also shown previously in a model of hypertension. Based on the data by Douglas et al., macrophage iNOS activity can be suppressed by specific deletion of macrophage GTPCH. Tie2-specific GTPCH deficiency had also functional/phenotypic consequences in the macrophages such as aggravated lipid accumulation and foam cell formation as well as impaired antioxidant defense mechanisms such as the absence of heme oxygenase-1 and peroxiredoxin-1 upregulation in GTPCH-deficient cells. By using bone marrow chimeras, the authors demonstrate that GTPCH deficiency in endothelial and myeloid (e.g. macrophage) cells is required for the aggravation of endothelial dysfunction and atherosclerosis, whereas the presence of only one