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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 5. — Effect of Gch1 knockdown on in vitro endothelial tube formation in endothelial cells. sEnd.1 murine endothelial cells (A–D) and primary human uterine microvascular endothelial cells (HutMECS; E and F) were transfected with a siRNA pool targeted to Gch1 or a nontargeting (nonspecific; NS) scrambled control siRNA. The cells were then harvested and analyzed for GTPCH (GTP cyclohydrolase 1) protein expression by Western blotting, or biopterin levels using high-performance liquid chromatography (HPLC) with electrochemical and fluorescent detection. A, Representative Western blot for GTPCH protein in sEND.1 mouse endothelial cell line treated with NS Gch1 siRNA or specific Gch1 siRNA (Gch1 siRNA). In sEND.1 treated with specific Gch1 siRNA, GTPCH protein was not detectable by Western blotting, whereas GTPCH was readily detected in sEND.1 treated with NS Gch1 siRNA. The blot is representative of four separate experiments. B, Intracellular tetrahydrobiopterin (BH4), dihydrobiopterin (BH2), total biopterins, and ratio of BH4 relative to oxidized biopterin species (BH4:BH2+B), measured by HPLC, were significantly reduced in Gch1-specific siRNA cells compared with NS siRNA cells (*P<0.05; n=4 per group). C, Representative photomicrographs of sEND.1 mouse endothelial cell treated with NS Gch1 siRNA or specific Gch1 siRNA plated on growth factor-reduced matrigel in the presence or absence of sepiapterin (Sep; 1 μM). Quantification of endothelial tube length and branching was performed by using Angiosys software and expressed in micrometers per field. D, In vitro endothelial tube formation and branching. Reduction of Gch1 expression in sEND.1 treated with specific Gch1 siRNA caused a marked decrease in endothelial cell growth and tubule formation compared to sEND.1 treated with NS Gch1 siRNA, which can be rescued by supplementation with Sep (*P<0.05; n=6 per group). The levels of BH4 in sEND.1 cells treated with NS Gch1 siRNA or specific Gch1 siRNA in the presence or absence of Sep (1 μM; *P<0.05; n=6 per group). E, Western blot analysis shows that cellular GTPCH protein was greatly reduced in HutMECs following exposure to GCH1-specific siRNA, compared to NS siRNA. The identity of bands obtained was confirmed using control lysates from human umbilical vein endothelial cells (HUVECs) treated with siRNA GCH1 (as negative control) or with NS siRNA (as positive control). GCH1 specific siRNA significantly reduced the detectable levels of the cellular biopterins and the ratio of BH4 to BH2+B (*P<0.05; n=4 per group). F, Representative photomicrographs of HutMECs treated with NS GCH1 siRNA or specific GCH1 siRNA plated on growth factor-reduced matrigel in the presence or absence of Sep (1 μM). Quantification of endothelial tube length and branching was performed by using Angiosys software and expressed in micrometers per field. In vitro endothelial tube formation and branching. Reduction of GCH1 expression in human uterine endothelial cells caused a significant decrease in endothelial cell growth and tubule formation, which can be rescued by supplementation with Sep (1 μM; *P<0.05; n=4 per group). B indicates biopterins.