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. 2021 Oct 25;78(6):1871–1884. doi: 10.1161/HYPERTENSIONAHA.120.17646

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© 2021 The Authors.

Hypertension is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited.

This article is made available via the PMC Open Access Subset for unrestricted re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the COVID-19 pandemic or until permissions are revoked in writing. Upon expiration of these permissions, PMC is granted a perpetual license to make this article available via PMC and Europe PMC, consistent with existing copyright protections.

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Figure 4. — GTPCH (GTP cyclohydrolase 1) protein, tetrahydrobiopterin (BH4) levels, NOS (NO synthase) activity, and in vitro endothelial tube formation in endothelial cells from normotensive and hypertensive pregnancies. A, Schematic diagram showing human umbilical vein endothelial cells (HUVECs) and placental extracellular vesicles were isolated from umbilical cords of placentas and from perfused placentas, respectively, from either normotensive (NT) or hypertensive pregnancies (HTP). B, High-performance liquid chromatography (HPLC) analysis of biopterins in HUVECs from NT and HTP. Biopterins are expressed per mg of cellular protein (*P<0.05; n=12–14 per group). C, Representative immunoblots showing GTPCH protein in HUVECs from NP and HTP pregnancies, with band density quantified relative to β-tubulin loading control (*P<0.05; n=9 per group). The identity of bands obtained was confirmed using control lysates from HUVECs treated with siRNA GCH1 (as negative control) or with nonspecific siRNA (as positive control). D, eNOS (endothelial NOS) activity was measured by conversion of ¹⁴C arginine in cell culture, followed by radiochemical HPLC quantification of ¹⁴C citrulline production. eNOS activity was greatly reduced in HUVECs from HTP compared with NT under basal conditions and when stimulated with calcium ionophore (A23187). Treatment with sepiapterin (Sep; 1 μM, converted to BH4 by the cellular pterin salvage pathway) significantly increased eNOS activity in both NT and HTP HUVECs, such that eNOS activity was no longer different between the groups. The nonselective NOS inhibitor NG-methyl-L-arginine (L-NMA), abolished eNOS activity in all groups (*P<0.05; n=6–8 per group). E, The levels of BH4 in HUVECs from NT and HTP treated with or without 1 μM Sep. Representative photomicrographs of HUVECs from NT and HTP plated on growth factor-reduced matrigel in the presence or absence of Sep (1 μM). E, Quantification of endothelial branches points and total tube length was performed using Angiosys software and expressed in micrometers per field. HUVECs from HTP showed lesser endothelial cell growth and tubule formation than HUVECs from NT but were rescued by supplementation with Sep (*P<0.05; n=6 per group). F, Extracellular vesicles were isolated by dual-lobe placental perfusion and ultracentrifugation from placentas from women with NT or from women with HTP. The levels of BH4, BH2, and total biopterins and ratio of BH4 relative to oxidized biopterin species (BH4:BH2+B) were measured by HPLC. BH4, total biopterins, and BH4/total biopterin ratio were significantly reduced in extracellular vesicles isolated from women with hypertensive pregnancies compared to women with normotensive pregnancies (*P<0.05; n=6 patients per group). B indicates biopterins.