Abstract 1493: Erythropoietin Stimulates Endothelial Biosynthesis of Tetrahydrobiopterin by Activation of Phosphatidylinositol 3-kinase/Protein Kinase B Signal Transduction Pathway
Erythropoietin (EPO) has been recognized as a tissue protective cytokine. Recently, it has been shown that vascular protective effects of EPO are dependent on activation of endothelial nitric oxide synthase (eNOS). Tetrahydrobiopterin (BH4) is an essential cofactor required for enzymatic activity of eNOS. Therefore, our objective was to characterize the effect of EPO on biosynthesis of BH4 in vascular wall. Incubation of isolated wild-type (C57BL/6J) mouse aortas for 18 hours at 37°C in minimal essential medium supplemented with recombinant human EPO (1–50 U/ml) caused concentration-dependent increase in intracellular levels of BH4 as determined by HPLC analysis. Maximum biosynthesis of BH4 was detected at therapeutic concentrations of 5 U/mL (15.8±1.3 pmol/mg protein; P<0.05 vs control: 8.2±0.4 pmol/mg protein; n=6 – 8). Oxidative products of BH4, 7,8-dihydrobiopterin, were unaffected by EPO indicating that EPO does not affect oxidation of BH4. Removal of the endothelium abolished EPO-induced biosynthesis of BH4 (P<0.05; n=5) demonstrating that the vascular endothelium is a major source of BH4. Treatment of intact isolated wild-type mouse aortas with a selective phosphatidylinositol (PI)3-kinase inhibitor wortmannin (1 μM) significantly reduced BH4 biosynthesis by EPO (8.4±0.6 pmol/mg protein; P<0.05; n=6). Stimulatory effect of EPO on production of BH4 in aorta was also detected in wild-type mice treated with recombinant human EPO (1000 U/kg, s.c. biweekly) for 14 days (P<0.05; n=6). This vascular effect was abolished in protein kinase B (Akt) 1-deficient mice treated with EPO (P<0.05; n=5). Furthermore, aortic GTP cyclohydrolase I (GTPCH I) enzymatic activity was augmented in EPO treated wild-type mice (0.48±0.12 pmol neopterin/mg protein; P<0.05 vs control: 0.23±0.05 pmol neopterin/mg; n=4) but not in EPO treated Akt 1-deficient mice (0.21±0.03 pmol neopterin/mg; n=4), indicating that the selective increase in BH4 levels was caused by de-novo biosynthesis of BH4 via Akt/GTPCH I pathway. Our results demonstrate that EPO stimulates biosynthesis of BH4 in vascular endothelium. This effect is most likely designed to provide optimal intracellular concentration of cofactor necessary for EPO-induced elevation of eNOS activity.