sepiapterin and Hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction in part as a result of enhanced oxidative stress. Function and survival of endothelial progenitor cells (EPCs, defined as sca1(+) c-kit(+) flk-1(+) bone marrow-derived cells), which significantly contribute to neovascularization and endothelial regeneration, depend on controlled production of reactive oxygen species (ROS). Mice heterozygous for the gene deletion of methylenetetrahydrofolate reductase (Mthfr(+/-)) have a 1.5- to 2-fold elevation in plasma homocysteine. This mild HHcy significantly reduced the number of circulating EPCs as well as their differentiation. Mthfr deficiency was also associated with increased ROS production and reduced nitric oxide (NO) generation in Mthfr(+/-) EPCs. Treatment of EPCs with sepiapterin, a precursor of tetrahydrobiopterin (BH(4)), a cofactor of endothelial nitric oxide synthase (eNOS), significantly reduced ROS and improved NO production. mRNA and protein expression of eNOS and the relative amount of eNOS dimer compared with monomer were decreased by Mthfr deficiency. Impaired differentiation of EPCs induced by Mthfr deficiency correlated with increased senescence, decreased telomere length, and reduced expression of SIRT1. Addition of sepiapterin maintained cell senescence and SIRT1 expression at levels comparable to the wild type. Taken together, these results demonstrate that Mthfr deficiency impairs EPC formation and increases EPC senescence by eNOS uncoupling and downregulation of SIRT1.

Topics: Animals; Cell Differentiation; Cellular Senescence; Down-Regulation; Endothelial Cells; Female; Homocystinuria; Hyperhomocysteinemia; Methylenetetrahydrofolate Reductase (NADPH2); Mice; Muscle Spasticity; Nitric Oxide; Nitric Oxide Synthase Type III; Oxidative Stress; Psychotic Disorders; Pterins; Reactive Oxygen Species; Sirtuin 1; Stem Cells; Telomere

A risk factor for cardiovascular disease, hyperhomocystinemia (HHcy), is associated with endothelial dysfunction. In this study, we examined the mechanistic role of HHcy in endothelial dysfunction.. Through the use of 2 functional models, aortic rings and intravital video microscopy of the cremaster, we found that arterial relaxation in response to the endothelium-dependent vessel relaxant, acetylcholine or the nitric oxide synthase (NOS) activator (A23187), was significantly impaired in cystathionine beta-synthase null (CBS(-/-)) mice. However, the vascular smooth muscle cell (VSMC) response to the nitric oxide (NO) donor (SNAP) was preserved in CBS(-/-) mice. In addition, superoxide dismutase and catalase failed to restore endothelium-dependent vasodilatation. Endothelial nitric oxide synthase (eNOS) activity was significantly reduced in mouse aortic endothelial cells (MAECs) of CBS(-/-) mice, as well as in Hcy-treated mouse and human aortic endothelial cells (HAECs). Hcy-mediated eNOS inhibition--which was not rescued by adenoviral transduction of superoxide dismutase and glutathione peroxidase, or by tetrahydrobiopterin, sepiapterin, and arginine supplementations in MAEC--was associated with decreased protein expression and increased threonine 495 phosphorylation of eNOS in HAECs. Ultimately, a protein kinase C (PKC) inhibitor, GF109203X (GFX), reversed Hcy-mediated eNOS inactivation and threonine 495 phosphorylation in HAECs.. These data suggest that HHcy impairs endothelial function and eNOS activity, primarily through PKC activation.

Topics: Animals; Antioxidants; Aorta, Thoracic; Arginine; Biopterins; Cells, Cultured; Cystathionine beta-Synthase; Endothelium, Vascular; Enzyme Activation; Female; Gene Expression Regulation, Enzymologic; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Homocysteine; Humans; Hyperhomocysteinemia; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Protein Kinase C; Pterins; Superoxide Dismutase; Vasodilation

Increased homocysteine is associated with the pregnancy complication preeclampsia and with later-life cardiovascular disease. Although elevated homocysteine persists after pregnancy, the vascular changes of preeclampsia abate with delivery, and cardiovascular disease occurs decades later. This suggests the vasculature during pregnancy may manifest increased sensitivity to homocysteine. We used the cystathionine-beta synthase (CBS)-deficient transgenic mouse to investigate whether hyperhomocysteinemia would differentially affect vascular function in nonpregnant and pregnant animals. Mesenteric arteries from nonpregnant and midpregnant (14 to 16 days) wild-type, heterozygous, and homozygous CBS-deficient transgenic mice were investigated for their response to vasoconstriction, endothelial-dependent, and endothelial-independent relaxation using an isometric wire myograph system. Endothelial-dependent vasodilation was similar in arteries from nonpregnant heterozygous and wild-type mice. In contrast, endothelial-dependent relaxation was reduced significantly in arteries from pregnant heterozygous animals compared with wild-type mice. Inhibition of NO synthesis blunted relaxation in arteries from pregnant wild-type but not pregnant heterozygous mice. Endothelial-dependent relaxation was restored by in vitro pretreatment with the tetrahydrobiopterin precursor sepiapterin. These data indicate that in pregnant mice, endothelial-dependent vasodilation is more sensitive to the effect of increased homocysteine than arteries from nonpregnant mice. This effect appears to result from a loss in NO-mediated relaxation that may be mediated by the oxidative inactivation of the NO synthase cofactor tetrahydrobiopterin.

Topics: Animals; Cystathionine beta-Synthase; Endothelium, Vascular; Female; Hyperhomocysteinemia; Mesenteric Arteries; Mice; Mice, Knockout; Mice, Transgenic; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oxidative Stress; Phenylephrine; Pregnancy; Pregnancy Complications; Pterins; Tyrosine; Vasodilation