Endothelial Cell Tetrahydrobiopterin Modulates Sensitivity to Ang (Angiotensin) II-Induced Vascular Remodeling, Blood Pressure, and Abdominal Aortic Aneurysm

Abstract

GTPCH (GTP cyclohydrolase 1, encoded by Gch1) is required for the synthesis of tetrahydrobiopterin; a critical regulator of endothelial NO synthase function. We have previously shown that mice with selective loss of Gch1 in endothelial cells have mild vascular dysfunction, but the consequences of endothelial cell tetrahydrobiopterin deficiency in vascular disease pathogenesis are unknown. We investigated the pathological consequence of Ang (angiotensin) II infusion in endothelial cell Gch1 deficient (Gch1^fl/fl Tie2cre) mice. Ang II (0.4 mg/kg per day, delivered by osmotic minipump) caused a significant decrease in circulating tetrahydrobiopterin levels in Gch1^fl/fl Tie2cre mice and a significant increase in the Nω-nitro-L-arginine methyl ester inhabitable production of H₂O₂ in the aorta. Chronic treatment with this subpressor dose of Ang II resulted in a significant increase in blood pressure only in Gch1^fl/fl Tie2cre mice. This finding was mirrored with acute administration of Ang II, where increased sensitivity to Ang II was observed at both pressor and subpressor doses. Chronic Ang II infusion in Gch1^fl/fl Tie2ce mice resulted in vascular dysfunction in resistance mesenteric arteries with an enhanced constrictor and decreased dilator response and medial hypertrophy. Altered vascular remodeling was also observed in the aorta with an increase in the incidence of abdominal aortic aneurysm formation in Gch1^fl/fl Tie2ce mice. These findings indicate a specific requirement for endothelial cell tetrahydrobiopterin in modulating the hemodynamic and structural changes induced by Ang II, through modulation of blood pressure, structural changes in resistance vessels, and aneurysm formation in the aorta.

Keywords: angiotensin II; aorta; blood pressure; endothelial cells; vascular remodeling.

Figure 1.

A, Representative immunoblots showing eNOS (endothelial NO synthase), nNOS (neuronal NOS), iNOS (inducible NOS), CD102 (endothelial cell marker), GTPCH (GTP cyclohydrolase I), and β-tubulin (loading control) proteins in whole aorta, media, and adventitia from wild-type (WT) and Gch1^fl/flTie2cre knockout (KO) mice (left). Quantitative data, measured as percentage band density of β-tubulin, showing GTPCH protein in whole aorta and media from WT and Gch1^fl/flTie2cre mice (right). B, HPLC analysis of biopterins in the whole aorta, media, and adventitia from WT and Gch1^fl/flTie2cre mice (*P<0.05 comparing genotype; #P<0.05 comparing treatment; n=6 animals per group). C, Aortic tetrahydrobiopterin (BH4), total biopterins, and BH4/BH2 ratio. D, Plasma BH4 and total biopterins levels (*P<0.05 comparing genotype; n=5–6 animals per group). E, Aortic nitrite/nitrate (NOx) levels and plasma NOx levels, from WT and Gch1^fl/flTie2cre mice infused with either saline or 0.4 mg/kg per day of Ang (angiotensin) II (*P<0.05 comparing genotype; n=5–6 animals per group). F, Aortic H₂O₂ production (expressed as the polyethylene glycol catalase inhibitable fraction) and aortic H₂O₂ production (expressed as the Nω-nitro-L-arginine methyl ester [L-NAME] inhibitable fraction) in WT and Gch1^fl/flTie2cre mice infused with either saline or Ang II (*P<0.05 comparing genotype; #P<0.05 comparing treatment; n=6 animals per group).

Figure 2.

Hemodynamic response to acute and chronic Ang (angiotensin) II stimulation in Gch1^fl/flTie2cre and wild-type (WT) mice. A, Representative continuous blood pressure (BP) traces, measured by Millar catheter, from WT and Gch1^fl/flTie2cre mice before and after a serial intraperitoneal bolus doses of Ang II (10–100 μg/kg) with quantitative data for change (Δ) in B systolic BP and heart rate after Ang II administration. Ang II caused a significantly greater increase in systolic BP in Gch1^fl/flTie2cre mice in a dose-dependent manner. Change in heart rate after Ang II administration, expressed in bpm (*P<0.05 comparing genotype; #P<0.05 comparing treatment; n=4–6 animals per group). C, Osmotic minipump containing Ang II (0.4 mg/kg per day) or saline was implanted in WT and Gch1^fl/flTie2cre mice. Systolic BP and heart rate, measured by noninvasive tail cuff, in WT and Gch1^fl/flTie2cre mice during 14 d of Ang II infusion. Systolic BP significantly increased in Gch1^fl/flTie2cre mice after 3 d of Ang II infusion but was unaltered in WT mice (*P<0.05 comparing treatment; †P<0.05 comparing genotype; n=5–6 animals per group). D, Heart rate significantly reduced in Gch1^fl/flTie2cre mice after Ang II infusion but unchanged in WT mice. E and F, Systolic BP and heart rate respectively in saline-infused WT and Gch1^fl/flTie2cre mice (n=3 animals per group).

Figure 3.

Impaired vasoconstrictions and endothelium-dependent vasodilatations in Gch1^fl/flTie2cre resistance mesenteric arteries in response to Ang (angiotensin) II stimulation. Isometric tension study of second-order resistance mesenteric arteries from wild-type (WT) and Gch1^fl/flTie2cre mice with either Ang II or saline infusion for 28 d. A, Vasoconstrictions in response to vasoconstrictor U46619 in mesenteric arteries. B, Maximum contraction (mN) in response to 1 µmol/L U46619. C, Endothelium-dependent vasodilatations in response to acetylcholine (ACh) in mesenteric arteries. D, Maximum relaxation (%) in response to 10 µmol/L ACh. E, Endothelium-independent vasodilatations in response to sodium nitroprusside (SNP) in mesenteric arteries. F, Maximum relaxation (%) in response to 10 µmol/L SNP (*P<0.05 comparing genotype; #P<0.05 comparing treatment; n=4–6 animals per group).

Figure 4.

Exacerbated resistance mesenteric artery remodeling in Gch1^fl/flTie2cre mice in response to Ang (angiotensin) II infusion. Vascular remodeling was analyzed in embed sections of the second-order branch of mesenteric arteries (perfusion fixed at 100 mm Hg) from wild-type (WT) and Gch1^fl/flTie2cre mice with either Ang II or saline infusion (0.4 mg/kg/ per day, 28 d). Representative images are shown (A) Masson–Goldner (MG) staining and (B) α-smooth muscle actin (α-SMA) staining of mesenteric arteries. Vascular remodeling was evaluated by (C) media thickness and (D) media area. Opened arrow indicates internal elastic lamina and closed arrow indicates external elastic lamina. (*P<0.05 comparing genotype; #P<0.05 comparing treatment; n=4–6 animals per group).

Figure 5.

Ang (angiotensin) II–induced vascular pathology of abdominal aortic aneurysm (AAA) in Gch1^fl/flTie2cre mice. Wild-type (WT) and Gch1^fl/flTie2cre mice were treated with a subpressor dose (0.4 mg/kg per day) of Ang II by osmotic minipump for 28 d. The lumen diameter of abdominal aortas was assessed in mice before and at the end of 28 d of Ang II infusion by ultrasound. A, In Ang II–infused mice, there was no significant difference in the diameter of abdominal aortas between WT and Gch1^fl/flTie2cre mice at day 0. After 28 d Ang II infusion, the lumen diameter of abdominal aorta of Gch1^fl/flTie2cre mice was significantly increased compared with WT controls. Ang II infusion caused a greater increase in aortic lumen diameter (expressed as changed in lumen diameter) in Gch1^fl/flTie2cre mice compared with WT mice (*P<0.05 comparing genotype; n=8–9 per group). B, In saline-infused mice, there was no significantly difference in the lumen diameter of abdominal aortas between WT and Gch1^fl/flTie2cre mice either at day 0 or day 28 of saline infusion (n=5–6 per group). C, Representative of aortic lumen diameter by ultrasound after Ang II infusion in WT and Gch1^fl/flTie2cre mice. D, Representative appearance of aortic dilation (opened arrow) and abdominal aortic aneurysm formation (yellow arrow). E, Event incidence of AAA pathology: dilation without AAA, AAA formation, and AAA rupture with sudden death in Ang II–infused Gch1^fl/flTie2cre and WT mice. F, Representative images of Masson–Goldner staining of abdominal aortic sections from WT and Gch1^fl/flTie2cre mice after saline or Ang II infusion for 28 d.